Modified clostridial toxins with altered targeting capabilities for clostridial toxin target cells

ABSTRACT

The specification discloses modified Clostridial toxins comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain and an enhanced Clostridial toxin binding domain; polynucleotide molecules encoding such modified Clostridial toxins; and method of producing such modified Clostridial toxins.

This is a continuation application and claims priority pursuant to 35U.S.C. §120 to U.S. patent application Ser. No. 11/817,937, filed Sep.6, 2007, now U.S. Pat. No. 8,052,979, a national stage patentapplication under 35 U.S.C. §371 of PCT application PCT/2006/009831filed on Mar. 14, 2006, which claims priority pursuant to 35 U.S.C.§19(e) to U.S. Provisional Patent Application Ser. No. 60/662,151 filedon Mar. 15, 2005 and U.S. Provisional Patent Application Ser. No.60/661,953 filed on Mar. 15, 2005, each incorporated entirely byreference.

The ability of Clostridial toxins, such as, e.g., Botulinum neurotoxins(BoNTs), BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F and BoNT/G, andTetanus neurotoxin (TeNT), to inhibit neuronal transmission are beingexploited in a wide variety of therapeutic and cosmetic applications,see e.g., William J. Lipham, COSMETIC AND CLINICAL APPLICATIONS OFBOTULINUM TOXIN (Slack, Inc., 2004). As an example, BOTOX® is currentlyapproved in one or more countries for the following indications:achalasia, adult spasticity, anal fissure, back pain, blepharospasm,bruxism, cervical dystonia, essential tremor, glabellar lines orhyperkinetic facial lines, headache, hemifacial spasm, hyperactivity ofbladder, hyperhidrosis, juvenile cerebral palsy, multiple sclerosis,myoclonic disorders, nasal labial lines, spasmodic dysphonia, strabismusand VII nerve disorder. In addition, Clostridial toxin therapies areproposed for treating neuromuscular disorders, see e.g., Kei Roger Aokiet al., Method for Treating Neuromuscular Disorders and Conditions withBotulinum Toxin Types A and B, U.S. Pat. No. 6,872,397 (Mar. 29, 2005);Rhett M. Schiffman, Methods for Treating Uterine Disorders, U.S. PatentPublication No. 2004/0175399 (Sep. 9, 2004); Richard L. Barron, Methodsfor Treating Ulcers and Gastroesophageal Reflux Disease, U.S. PatentPublication No. 2004/0086531 (May 7, 2004); and Kei Roger Aoki, et al.,Method for Treating Dystonia with Botulinum Toxin C to G, U.S. Pat. No.6,319,505 (Nov. 20, 2001); eye disorders, see e.g., Eric R. First,Methods and Compositions for Treating Eye Disorders, U.S. PatentPublication No. 2004/0234532 (Nov. 25, 2004); Kei Roger Aoki et al.,Botulinum Toxin Treatment for Blepharospasm, U.S. Patent Publication No.2004/0151740 (Aug. 5, 2004); and Kei Roger Aoki et al., Botulinum ToxinTreatment for Strabismus, U.S. Patent Publication No. 2004/0126396 (Jul.1, 2004); pain, see e.g., Kei Roger Aoki et al., Pain Treatment byPeripheral Administration of a Neurotoxin, U.S. Pat. No. 6,869,610 (Mar.22, 2005); Stephen Donovan, Clostridial Toxin Derivatives and Methods toTreat Pain, U.S. Pat. No. 6,641,820 (Nov. 4, 2003); Kei Roger Aoki, etal., Method for Treating Pain by Peripheral Administration of aNeurotoxin, U.S. Pat. No. 6,464,986 (Oct. 15, 2002); Kei Roger Aoki andMinglei Cui, Methods for Treating Pain, U.S. Pat. No. 6,113,915 (Sep. 5,2000); Martin A. Voet, Methods for Treating Fibromyalgia, U.S. Pat. No.6,623,742 (Sep. 23, 2003); Martin A. Voet, Botulinum Toxin Therapy forFibromyalgia, U.S. Patent Publication No. 2004/0062776 (Apr. 1, 2004);and Kei Roger Aoki et al., Botulinum Toxin Therapy for Lower Back Pain,U.S. Patent Publication No. 2004/0037852 (Feb. 26, 2004); muscleinjuries, see e.g., Gregory F. Brooks, Methods for Treating MuscleInjuries, U.S. Pat. No. 6,423,319 (Jul. 23, 2002); headache, see e.g.,Martin Voet, Methods for Treating Sinus Headache, U.S. Pat. No.6,838,434 (Jan. 4, 2005); Kei Roger Aoki et al., Methods for TreatingTension Headache, U.S. Pat. No. 6,776,992 (Aug. 17, 2004); and Kei RogerAoki et al., Method for Treating Headache, U.S. Pat. No. 6,458,365 (Oct.1, 2002); William J. Binder, Method for Reduction of Migraine HeadachePain, U.S. Pat. No. 5,714,469 (Feb. 3, 1998); cardiovascular diseases,see e.g., Gregory F. Brooks and Stephen Donovan, Methods for TreatingCardiovascular Diseases with Botulinum Toxin, U.S. Pat. No. 6,767,544(Jul. 27, 2004); neurological disorders, see e.g., Stephen Donovan,Parkinson's Disease Treatment, U.S. Pat. No. 6,620,415 (Sep. 16, 2003);and Stephen Donovan, Method for Treating Parkinson's Disease with aBotulinum Toxin, U.S. Pat. No. 6,306,403 (Oct. 23, 2001);neuropsychiatric disorders, see e.g., Stephen Donovan, Botulinum ToxinTherapy for Neuropsychiatric Disorders, U.S. Patent Publication No.2004/0180061 (Sep. 16, 2004); and Steven Donovan, Therapeutic Treatmentsfor Neuropsychiatric Disorders, U.S. Patent Publication No. 2003/0211121(Nov. 13, 2003); endocrine disorders, see e.g., Stephen Donovan, Methodfor Treating Endocrine Disorders, U.S. Pat. No. 6,827,931 (Dec. 7,2004); Stephen Donovan, Method for Treating Thyroid Disorders with aBotulinum Toxin, U.S. Pat. No. 6,740,321 (May 25, 2004); Kei Roger Aokiet al., Method for Treating a Cholinergic Influenced Sweat Gland, U.S.Pat. No. 6,683,049 (Jan. 27, 2004); Stephen Donovan, Neurotoxin Therapyfor Diabetes, U.S. Pat. No. 6,416,765 (Jul. 9, 2002); Stephen Donovan,Methods for Treating Diabetes, U.S. Pat. No. 6,337,075 (Jan. 8, 2002);Stephen Donovan, Method for Treating a Pancreatic Disorder with aNeurotoxin, U.S. Pat. No. 6,261,572 (Jul. 17, 2001); Stephen Donovan,Methods for Treating Pancreatic Disorders, U.S. Pat. No. 6,143,306 (Nov.7, 2000); cancers, see e.g., Stephen Donovan, Methods for Treating BoneTumors, U.S. Pat. No. 6,565,870 (May 20, 2003); Stephen Donovan, Methodfor Treating Cancer with a Neurotoxin to Improve Patient Function, U.S.Pat. No. 6,368,605 (Apr. 9, 2002); Stephen Donovan, Method for TreatingCancer with a Neurotoxin, U.S. Pat. No. 6,139,845 (Oct. 31, 2000); andMitchell F. Brin and Stephen Donovan, Methods for Treating DiverseCancers, U.S. Patent Publication No. 2005/0031648 (Feb. 10, 2005); oticdisorders, see e.g., Stephen Donovan, Neurotoxin Therapy for Inner EarDisorders, U.S. Pat. No. 6,358,926 (Mar. 19, 2002); and Stephen Donovan,Method for Treating Otic Disorders, U.S. Pat. No. 6,265,379 (Jul. 24,2001); autonomic disorders, see, e.g., Pankai J. Pasricha and Anthony N.Kalloo, Method for Treating Gastrointestinal Muscle Disorders and OtherSmooth Muscle Dysfunction, U.S. Pat. No. 5,437,291 (Aug. 1, 1995); aswell as other disorders, see e.g., William J. Binder, Method forTreatment of Skin Lesions Associated with Cutaneous Cell-proliferativeDisorders, U.S. Pat. No. 5,670,484 (Sep. 23, 1997); Eric R. First,Application of Botulinum Toxin to the Management of NeurogenicInflammatory Disorders, U.S. Pat. No. 6,063,768 (May 16, 2000); MarvinSchwartz and Brian J. Freund, Method to Reduce Hair Loss and StimulateHair Growth, U.S. Pat. No. 6,299,893 (Oct. 9, 2001); Jean D. A.Carruthers and Alastair Carruthers, Cosmetic Use of Botulinum Toxin forTreatment of Downturned Mouth, U.S. Pat. No. 6,358,917 (Mar. 19, 2002);Stephen Donovan, Use of a Clostridial Toxin to Reduce Appetite, U.S.Patent Publication No. 2004/40253274 (Dec. 16, 2004); and Howard I. Katzand Andrew M. Blumenfeld, Botulinum Toxin Dental Therapies andProcedures, U.S. Patent Publication No. 2004/0115139 (Jun. 17, 2004);Kei Roger Aoki, et al., Treatment of Neuromuscular Disorders andConditions with Different Botulinum, U.S. Patent Publication No.2002/0010138 (Jan. 24, 2002); and Kei Roger Aoki, et al., Use ofBotulinum Toxins for Treating Various Disorders and Conditions andAssociated Pain, U.S. Patent Publication No. 2004/0013692 (Jan. 22,2004). In addition, the expected use of Clostridial toxins, such as,e.g., BoNTs and TeNT, in therapeutic and cosmetic treatments of humansand other mammals is anticipated to expand to an ever widening range ofdiseases and ailments that can benefit from the properties of thesetoxins.

Clostridial toxin therapies are successfully used for many indications.Generally, administration of a Clostridial toxin treatment is welltolerated. However, toxin administration in some applications can bechallenging because of the larger doses required to achieve a beneficialeffect. Larger doses can increase the likelihood that the toxin may movethrough the interstitial fluids and the circulatory systems, such as,e.g., the cardiovascular system and the lymphatic system, of the body,resulting in the undesirable dispersal of the toxin to areas nottargeted for toxin treatment. Such dispersal can lead to undesirableside effects, such as, e.g., inhibition of neurotransmitter release inneurons not targeted for treatment or paralysis of a muscle not targetedfor treatment. For example, a patient administered a therapeuticallyeffective amount of a BoNT/A treatment into the neck muscles fortorticollis may develop dysphagia because of dispersal of the toxin intothe oropharynx. Thus, there remains a need for improved Clostridialtoxins that are effective at the site of treatment, but have negligibleto minimal effects in areas not targeted for a toxin treatment.

The growing clinical, therapeutic and cosmetic use of Clostridial toxinsin therapies requiring larger doses necessitates the pharmaceuticalindustry to develop modified Clostridial toxins that are effective atthe target site of the application, but reduce or prevent theundesirable side-effects associated with the dispersal of the toxins toan unwanted location or locations. The present invention provides novelClostridial toxins that reduce or prevent unwanted side-effectsassociated with toxin dispersal into non-targeted areas. These andrelated advantages are useful for various clinical, therapeutic andcosmetic applications, such as, e.g., the treatment of neuromusculardisorders, neuropathic disorders, eye disorders, pain, muscle injuries,headache, cardiovascular diseases, neuropsychiatric disorders, endocrinedisorders, cancers, otic disorders and hyperkinetic facial lines, aswell as, other disorders where a Clostridial toxin administration to amammal can produce a beneficial effect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of the current paradigm of neurotransmitterrelease and Clostridial toxin intoxication in a central and peripheralneuron. FIG. 1A shows a schematic for the neurotransmitter releasemechanism of a central and peripheral neuron. The release process can bedescribed as comprising two steps: 1) vesicle docking, where thevesicle-bound SNARE protein of a vesicle containing neurotransmittermolecules associates with the membrane-bound SNARE proteins located atthe plasma membrane; and 2) neurotransmitter release, where the vesiclefuses with the plasma membrane and the neurotransmitter molecules areexocytosed. FIG. 1B shows a schematic of the intoxication mechanism fortetanus and botulinum toxin activity in a central and peripheral neuron.This intoxication process can be described as comprising four steps: 1)receptor binding, where a Clostridial toxin binds to a Clostridialreceptor system and initiates the intoxication process; 2) complexinternalization, where after toxin binding, a vesicle containing thetoxin/receptor system complex is endocytosed into the cell; 3) lightchain translocation, where multiple events are thought to occur,including, e.g., changes in the internal pH of the vesicle, formation ofa channel pore comprising the H_(N) domain of the Clostridial toxinheavy chain, separation of the Clostridial toxin light chain from theheavy chain, and release of the active light chain and 4) enzymatictarget modification, where the activate light chain of Clostridial toxinproteolytically cleaves its target SNARE substrate, such as, e.g.,SNAP-25, VAMP or Syntaxin, thereby preventing vesicle docking andneurotransmitter release.

FIG. 2 shows the domain organization of naturally-occurring Clostridialtoxins. The single chain form depicts the amino to carboxyl linearorganization comprising an an enzymatic domain, a translocation domain,and a binding domain. The di-chain loop region located between thetranslocation and enzymatic domains is depicted by the double SSbracket. This region comprises an endogenous di-chain loop proteasecleavage site that upon proteolytic cleavage with a naturally-occurringprotease, such as, e.g., an endogenous Clostridial toxin protease or anaturally-occurring protease produced in the environment, converts thesingle chain form of the toxin into the di-chain form. Above thesingle-chain form, the H_(CC) region of the Clostridial toxin bindingdomain is depicted. This region comprises the β-trefoil domain whichcomprises in a amino to carboxyl linear organization an α-fold, a β4/β5hairpin turn, a β-fold, a β8/β9 hairpin turn and a γ-fold.

FIG. 3 shows modified Clostridial toxins with an enhanced targetingdomain located at the amino terminus of the modified toxin. FIG. 3Adepicts the single polypeptide form of a modified Clostridial toxin withan amino to carboxyl linear organization comprising an enhancedtargeting domain, an enzymatic domain and a translocation domain, withthe di-chain loop region depicted by the double SS bracket. Aproteolytic cleavage site (P) within a di-chain loop region is locatedbetween the translocation and enzymatic domains. Upon proteolyticcleavage with a P protease, the single chain form of the toxin isconverted to the di-chain form. The P protease site can be a Clostridialtoxin endogenous protease cleavage site or a non-Clostridial toxinexogenous protease cleavage site. Spacers can be placed between thetargeting and translocation domains, the translocation and enzymaticdomains or any combination thereof. FIG. 3B depicts the singlepolypeptide form of a modified Clostridial toxin with an amino tocarboxyl linear organization comprising an enhanced targeting domain, anenzymatic domain and a translocation domain, with the di-chain loopregion depicted by the double SS bracket. A proteolytic cleavage site(P) within a di-chain loop region is located between the enzymatic andtranslocation domains. Upon proteolytic cleavage with a P protease, thesingle chain form of the toxin is converted to the di-chain form. The Pprotease site can be a Clostridial toxin endogenous protease cleavagesite or a non-Clostridial toxin exogenous protease cleavage site.Spacers can be placed between the targeting and enzymatic domains, theenzymatic and translocation domains or any combination thereof.

FIG. 4 shows modified Clostridial toxins with an enhanced targetingdomain located between the other two domains. FIG. 4A depicts the singlepolypeptide form of a modified Clostridial toxin with an amino tocarboxyl linear organization comprising an enzymatic domain, an enhancedtargeting domain and a translocation domain, with the di-chain loopregion depicted by the double SS bracket. A proteolytic cleavage site(P) within a di-chain loop region is located between the enzymatic andtargeting domains. Upon proteolytic cleavage with a P protease, thesingle chain form of the toxin is converted to the di-chain form. The Pprotease site can be a Clostridial toxin endogenous protease cleavagesite or a non-Clostridial toxin exogenous protease cleavage site.Spacers can be placed between the enzymatic and targeting domains, thetargeting and translocation domains or any combination thereof. FIG. 4Bdepicts the single polypeptide form of a modified Clostridial toxin withan amino to carboxyl linear organization comprising a translocationdomain, an enhanced targeting domain and an enzymatic domain, with thedi-chain loop region depicted by the double SS bracket. A proteolyticcleavage site (P) within a di-chain loop region is located between thetranslocation and targeting domains. Upon proteolytic cleavage with a Pprotease, the single chain form of the toxin is converted to thedi-chain form. The P protease site can be a Clostridial toxin endogenousprotease cleavage site or a non-Clostridial toxin exogenous proteasecleavage site. Spacers can be placed between the translocation andtargeting domains, the targeting and enzymatic domains or anycombination thereof.

FIG. 5 shows modified Clostridial toxins with an enhanced targetingdomain located at the carboxyl terminus of the modified toxin. FIG. 5Adepicts the single polypeptide form of a modified Clostridial toxin withan amino to carboxyl linear organization comprising an enzymatic domain,a translocation domain and an enhanced targeting domain, with thedi-chain loop region depicted by the double SS bracket. A proteolyticcleavage site (P) within a di-chain loop region is located between theenzymatic and translocation domains. Upon proteolytic cleavage with a Pprotease, the single chain form of the toxin is converted to thedi-chain form. The P protease site can be a Clostridial toxin endogenousprotease cleavage site or a non-Clostridial toxin exogenous proteasecleavage site. Spacers can be placed between the enzymatic andtranslocation domains, the translocation and targeting domains or anycombination thereof. FIG. 5B depicts the single polypeptide form of amodified Clostridial toxin with an amino to carboxyl linear organizationcomprising a translocation domain, an enzymatic domain and an enhancedtargeting domain, with the di-chain loop region depicted by the doubleSS bracket. A proteolytic cleavage site (P) within a di-chain loopregion is located between the translocation and enzymatic domains. Uponproteolytic cleavage with a P protease, the single chain form of thetoxin is converted to the di-chain form. The P protease site can be aClostridial toxin endogenous protease cleavage site or a non-Clostridialtoxin exogenous protease cleavage site. Spacers can be placed betweenthe translocation and enzymatic domains, the enzymatic and targetingdomains or any combination thereof.

DETAILED DESCRIPTION

The present invention discloses modified Clostridial toxins that exhibitenhanced binding activity for cells targeted by naturally-occurringClostridial toxins. Enhanced binding activity is achieved by replacing anaturally-occurring binding domain of a Clostridial toxin with a bindingdomain for a non-Clostridial toxin receptor system present on aClostridial toxin target cell. This enhanced binding activity for atarget cell should allow lower effective doses of a modified Clostridialtoxin to be administered to an individual because more toxin will bedelivered to a target cell. Thus modified Clostridial toxins withenhanced binding activity will reduce the undesirable dispersal of thetoxin to areas not targeted for treatment, thereby reducing orpreventing the undesirable side-effects associated with diffusion of aClostridial toxin to an unwanted location.

Aspects of the present invention provide modified Clostridial toxinscomprising a Clostridial toxin enzymatic domain, a Clostridial toxintranslocation domain and an enhanced targeting domain, wherein themodified Clostridial toxin exhibits a binding activity for anon-Clostridial toxin receptor system present on a Clostridial toxintarget cell relative to a naturally-occurring Clostridial toxin. It isenvisioned that the location of the enhanced targeting domain in themodified Clostridial toxins of the present specification can be locatedat the amino terminus of the toxin, between the enzymatic andtranslocation domains or at the carboxyl terminus of the toxin. Thus, amodified Clostridial toxins disclosed in the present specification cancomprise an amino to carboxyl domain arrangement of, e.g., an enhancedtargeting domain, a Clostridial toxin translocation domain and aClostridial toxin enzymatic domain; an enhanced targeting domain, aClostridial toxin enzymatic domain and a Clostridial toxin translocationdomain; a Clostridial toxin enzymatic domain, an enhanced targetingdomain and a Clostridial toxin translocation domain; a Clostridial toxintranslocation domain, an enhanced targeting domain and a Clostridialtoxin enzymatic domain; a Clostridial toxin enzymatic domain, aClostridial toxin translocation domain and an enhanced targeting domain;and a Clostridial toxin translocation domain, a Clostridial toxinenzymatic domain and an enhanced targeting domain.

Other aspects of the present invention provide polynucleotide moleculesencoding modified Clostridial toxins comprising a Clostridial toxinenzymatic domain, a Clostridial toxin translocation domain and anenhanced targeting domain, wherein the modified Clostridial toxinexhibits an enhanced binding activity for a non-Clostridial toxinreceptor system present on a Clostridial toxin target cell relative to anaturally-occurring Clostridial toxin. It is envisioned that thelocation of the enhanced targeting domain of the modified Clostridialtoxins encoded by polynucleotide molecules of the present specificationcan be located at the amino terminus of the toxin, between the enzymaticand translocation domains or at the carboxyl terminus of the toxin.Thus, polynucleotide molecules disclosed in the present specificationcan encode modified Clostridial toxins comprising an amino to carboxyldomain arrangement of, e.g., an enhanced targeting domain, a Clostridialtoxin translocation domain and a Clostridial toxin enzymatic domain; anenhanced targeting domain, a Clostridial toxin enzymatic domain and aClostridial toxin translocation domain; a Clostridial toxin enzymaticdomain, an enhanced targeting domain and a Clostridial toxintranslocation domain; a Clostridial toxin translocation domain, anenhanced targeting domain and a Clostridial toxin enzymatic domain; aClostridial toxin enzymatic domain, a Clostridial toxin translocationdomain and an enhanced targeting domain; and a Clostridial toxintranslocation domain, a Clostridial toxin enzymatic domain and anenhanced targeting domain.

Other aspects of the present invention provide methods of producing amodified Clostridial toxin disclosed in the present specification, themethod comprising the step of expressing in a cell a polynucleotidemolecule encoding a modified Clostridial toxin comprising a Clostridialtoxin enzymatic domain, a Clostridial toxin translocation domain and anenhanced targeting domain, wherein the modified Clostridial toxinexhibits an enhanced targeting activity for a Clostridial toxin targetcell relative to a naturally-occurring Clostridial toxin. Other aspectsof the present invention provide methods of producing a modifiedClostridial toxin disclosed in the present specification, the methodcomprising the steps of introducing in a cell an expression constructcomprising a polynucleotide molecule encoding a modified Clostridialtoxin comprising a Clostridial toxin enzymatic domain, a Clostridialtoxin translocation domain and an enhanced targeting domain, wherein themodified Clostridial toxin exhibits an enhanced targeting activity for aClostridial toxin target cell relative to a naturally-occurringClostridial toxin and expressing the expression construct in the cell.

The toxins produced by Clostridium botulinum, Clostridium tetani,Clostridium baratii and Clostridium butyricum are the most widely usedin therapeutic and cosmetic treatments of humans and other mammals.Strains of C. botulinum produce seven antigenically-distinct types ofBotulinum toxins (BoNTs), which have been identified by investigatingbotulism outbreaks in man (BoNT/A, /B, /E and /F), animals (BoNT/C1 and/D), or isolated from soil (BoNT/G). While all seven botulinum toxins(BoNT) serotypes have similar structure and pharmacological properties,each also displays heterogeneous bacteriological characteristics. Incontrast, tetanus toxin (TeNT) is produced by a uniform group of C.tetani. Two other species of Clostridia, C. baratii and C. butyricum,also produce toxins similar to BoNT/F and BoNT/E, respectively.

Clostridia toxins possess approximately 35% amino acid identity witheach other and share the same functional domain organization and overallstructural architecture. Clostridial toxins are each translated as asingle chain polypeptide of approximately 150 kDa that is subsequentlycleaved by proteolytic scission within a disulfide loop by anaturally-occurring protease, such as, e.g., an endogenous Clostridialtoxin protease or a naturally-occurring proteases produced in theenvironment (FIG. 2). This posttranslational processing yields adi-chain molecule comprising an approximately 50 kDa light chain (LC)and an approximately 100 kDa heavy chain (HC) held together by a singledisulfide bond and noncovalent interactions. Each mature di-chainmolecule comprises three functionally distinct domains: 1) an enzymaticdomain located in the LC that includes a metalloprotease regioncontaining a zinc-dependent endopeptidase activity which specificallytargets core components of the neurotransmitter release apparatus (Table1); 2) a translocation domain contained within the amino-terminal halfof the HC (H_(N)) that facilitates release of the LC from intracellularvesicles into the cytoplasm of the target cell (Table 1); and 3) abinding domain found within the carboxyl-terminal half of the HC (H_(C))that determines the binding activity and binding specificity of thetoxin to the receptor complex located at the surface of the target cell(Table 1).

TABLE 1 Clostridial Toxin Reference Sequences and Regions Toxin SEQ IDNO: LC H_(N) H_(C) BoNT/A 1 M1-K448 A449-K871 N872-L1296 BoNT/B 2M1-K441 A442-S858 E859-E1291 BoNT/C1 3 M1-K449 T450-N866 N867-E1291BoNT/D 4 M1-R445 D446-N862 S863-E1276 BoNT/E 5 M1-R422 K423-K845R846-K1252 BoNT/F 6 M1-K439 A440-K864 K865-E1274 BoNT/G 7 M1-K446S447-S863 N864-E1297 TeNT 8 M1-A457 S458-V879 I880-D1315

The binding, translocation and enzymatic activity of these threefunctional domains are all necessary for toxicity. While all details ofthis process are not yet precisely known, the overall cellularintoxication mechanism whereby Clostridial toxins enter a neuron andinhibit neurotransmitter release is similar, regardless of serotype orsubtype. Although the applicants have no wish to be limited by thefollowing description, the intoxication mechanism can be described ascomprising at least four steps: 1) receptor binding, 2) complexinternalization, 3) light chain translocation, and 4) enzymatic targetmodification (see FIG. 1). The process is initiated when the H_(C)domain of a Clostridial toxin binds to a toxin-specific receptor systemlocated on the plasma membrane surface of a target cell. The bindingspecificity of a receptor complex is thought to be achieved, in part, byspecific combinations of gangliosides and protein receptors that appearto distinctly comprise each Clostridial toxin receptor complex. Oncebound, the toxin/receptor complexes are internalized by endocytosis andthe internalized vesicles are sorted to specific intracellular routes.The translocation step appears to be triggered by the acidification ofthe vesicle compartment. This process seems to initiate two importantpH-dependent structural rearrangements that increase hydrophobicity andpromote formation di-chain form of the toxin. Once activated, lightchain endopeptidase of the toxin is released from the intracellularvesicle into the cytosol where it appears to specifically targets one ofthree known core components of the neurotransmitter release apparatus.These core proteins, vesicle-associated membrane protein(VAMP)/synaptobrevin, synaptosomal-associated protein of 25 kDa(SNAP-25) and Syntaxin, are necessary for synaptic vesicle docking andfusion at the nerve terminal and constitute members of the solubleN-ethylmaleimide-sensitive factor-attachment protein-receptor (SNARE)family. BoNT/A and BoNT/E cleave SNAP-25 in the carboxyl-terminalregion, releasing a nine or twenty-six amino acid segment, respectively,and BoNT/C1 also cleaves SNAP-25 near the carboxyl-terminus. Thebotulinum serotypes BoNT/B, BoNT/D, BoNT/F and BoNT/G, and tetanustoxin, act on the conserved central portion of VAMP, and release theamino-terminal portion of VAMP into the cytosol. BoNT/C1 cleavessyntaxin at a single site near the cytosolic membrane surface. Theselective proteolysis of synaptic SNAREs accounts for the block ofneurotransmitter release caused by Clostridial toxins in vivo. The SNAREprotein targets of Clostridial toxins are common to exocytosis in avariety of non-neuronal types; in these cells, as in neurons, lightchain peptidase activity inhibits exocytosis, see, e.g., Yann Humeau etal., How Botulinum and Tetanus Neurotoxins Block NeurotransmitterRelease, 82(5) Biochimie. 427-446 (2000); Kathryn Turton et al.,Botulinum and Tetanus Neurotoxins: Structure, Function and TherapeuticUtility, 27(11) Trends Biochem. Sci. 552-558. (2002); Giovanna Lalli etal., The Journey of Tetanus and Botulinum Neurotoxins in Neurons, 11(9)Trends Microbiol. 431-437, (2003).

Aspects of the present invention provide, in part, a modifiedClostridial toxin. As used herein, the term “modified Clostridial toxin”means any polypeptide that can execute the overall cellular mechanismwhereby a Clostridial toxin enters a neuron and inhibitsneurotransmitter release and encompasses the binding of a Clostridialtoxin to a low or high affinity receptor complex, the internalization ofthe toxin, the translocation of the Clostridial toxin light chain intothe cytoplasm and the enzymatic modification of a Clostridial toxinsubstrate. A modified Clostridial toxin disclosed in the presentspecification is distinguished from a naturally-occurring Clostridialtoxin by the fact that a modified Clostridial toxin lacks the cellbinding activity of a naturally-occurring binding domain found in aClostridial toxin. Instead, a modified Clostridial toxin disclosed inthe present specification comprises an enhanced targeting domain thatdetermines the binding activity of the modified Clostridial toxin to anendogenous Clostridial toxin receptor system located at the surface ofthe target cell. By definition, a naturally-occurring Clostridial toxinlacks an enhanced targeting domain.

Aspects of the present invention provide, in part, a Clostridial toxinenzymatic domain. As used herein, the term “Clostridial toxin enzymaticdomain” means any Clostridial toxin polypeptide that can execute theenzymatic target modification step of the intoxication process. Thus, aClostridial toxin enzymatic domain specifically targets a Clostridialtoxin substrate and encompasses the proteolytic cleavage of aClostridial toxin substrate, such as, e.g., SNARE proteins like aSNAP-25 substrate, a VAMP substrate and a Syntaxin substrate.Non-limiting examples of a Clostridial toxin enzymatic domain include,e.g., a Clostridial toxin light chain region such as, e.g., a BoNT/Alight chain region, a BoNT/B light chain region, a BoNT/C1 light chainregion, a BoNT/D light chain region, a BoNT/E light chain region, aBoNT/F light chain region, a BoNT/G light chain region, and a TeNT lightchain region.

A Clostridial toxin enzymatic domain includes, without limitation,naturally occurring Clostridial toxin light chain variants, such as,e.g., Clostridial toxin light chain isoforms and Clostridial toxin lightchain subtypes; non-naturally occurring Clostridial toxin light chainvariants, such as, e.g., conservative Clostridial toxin light chainvariants, non-conservative Clostridial toxin light chain variants,Clostridial toxin light chain chimerics, active Clostridial toxin lightchain fragments thereof, or any combination thereof.

As used herein, the term “Clostridial toxin light chain variant,”whether naturally-occurring or non-naturally-occurring, means aClostridial toxin light chain that has at least one amino acid changefrom the corresponding region of the disclosed reference sequences (seeTable 1) and can be described in percent identity to the correspondingregion of that reference sequence. Unless expressly indicated, allClostridial toxin light chain variants disclosed in the presentspecification are capable of executing the enzymatic target modificationstep of the intoxication process. As non-limiting examples, a BoNT/Alight chain variant comprising amino acids 1-448 of SEQ ID NO: 1 willhave at least one amino acid difference, such as, e.g., an amino acidsubstitution, deletion or addition, as compared to the amino acid region1-448 of SEQ ID NO: 1; a BoNT/B light chain variant comprising aminoacids 1-441 of SEQ ID NO: 2 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the amino acid region 1-441 of SEQ ID NO: 2; aBoNT/C1 light chain variant comprising amino acids 1-449 of SEQ ID NO: 3will have at least one amino acid difference, such as, e.g., an aminoacid substitution, deletion or addition, as compared to the amino acidregion 1-449 of SEQ ID NO: 3; a BoNT/D light chain variant comprisingamino acids 1-445 of SEQ ID NO: 4 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the amino acid region 1-445 of SEQ ID NO: 4; aBoNT/E light chain variant comprising amino acids 1-422 of SEQ ID NO: 5will have at least one amino acid difference, such as, e.g., an aminoacid substitution, deletion or addition, as compared to the amino acidregion 1-422 of SEQ ID NO: 5; a BoNT/F light chain variant comprisingamino acids 1-439 of SEQ ID NO: 6 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the amino acid region 1-439 of SEQ ID NO: 6; aBoNT/G light chain variant comprising amino acids 1-446 of SEQ ID NO: 7will have at least one amino acid difference, such as, e.g., an aminoacid substitution, deletion or addition, as compared to the amino acidregion 1-446 of SEQ ID NO: 7; and a TeNT light chain variant comprisingamino acids 1-457 of SEQ ID NO: 8 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the amino acid region 1-457 of SEQ ID NO: 8.

It is recognized by those of skill in the art that within each serotypeof Clostridial toxin there can be naturally occurring Clostridial toxinlight chain variants that differ somewhat in their amino acid sequence,and also in the nucleic acids encoding these proteins. For example,there are presently four BoNT/A subtypes, BoNT/A1, BoNT/A2, BoNT/A3 andBoNT/A4, with specific light chain subtypes showing approximately 95%amino acid identity when compared to another BoNT/A light chain subtype.As used herein, the term “naturally occurring Clostridial toxin lightchain variant” means any Clostridial toxin light chain produced by anaturally-occurring process, including, without limitation, Clostridialtoxin light chain isoforms produced from alternatively-splicedtranscripts, Clostridial toxin light chain isoforms produced byspontaneous mutation and Clostridial toxin light chain subtypes. Anaturally occurring Clostridial toxin light chain variant can functionin substantially the same manner as the reference Clostridial toxinlight chain on which the naturally occurring Clostridial toxin lightchain variant is based, and can be substituted for the referenceClostridial toxin light chain in any aspect of the present invention. Anaturally occurring Clostridial toxin light chain variant may substituteone or more amino acids, two or more amino acids, three or more aminoacids, four or more amino acids, five or more amino acids, ten or moreamino acids, 20 or more amino acids, 30 or more amino acids, 40 or moreamino acids, 50 or more amino acids or 100 or more amino acids from thereference Clostridial toxin light chain on which the naturally occurringClostridial toxin light chain variant is based. A naturally occurringClostridial toxin light chain variant can also substitute at least 10contiguous amino acids, at least 15 contiguous amino acids, at least 20contiguous amino acids, or at least 25 contiguous amino acids from thereference Clostridial toxin light chain on which the naturally occurringClostridial toxin light chain variant is based, that possess at least50% amino acid identity, 65% amino acid identity, 75% amino acididentity, 85% amino acid identity or 95% amino acid identity to thereference Clostridial toxin light chain on which the naturally occurringClostridial toxin light chain variant is based.

A non-limiting examples of a naturally occurring Clostridial toxin lightchain variant is a Clostridial toxin light chain isoform such as, e.g.,a BoNT/A light chain isoform, a BoNT/B light chain isoform, a BoNT/C1light chain isoform, a BoNT/D light chain isoform, a BoNT/E light chainisoform, a BoNT/F light chain isoform, a BoNT/G light chain isoform, anda TeNT light chain isoform. A Clostridial toxin light chain isoform canfunction in substantially the same manner as the reference Clostridialtoxin light chain on which the Clostridial toxin light chain isoform isbased, and can be substituted for the reference Clostridial toxin lightchain in any aspect of the present invention.

Another non-limiting examples of a naturally occurring Clostridial toxinlight chain variant is a Clostridial toxin light chain subtype such as,e.g., a light chain from subtype BoNT/A1, BoNT/A2, BoNT/A3 and BoNT/A4;a light chain from subtype BoNT/B1, BoNT/B2, BoNT/B bivalent and BoNT/Bnonproteolytic; a light chain from subtype BoNT/C1-1 and BoNT/C1-2; alight chain from subtype BoNT/E1, BoNT/E2 and BoNT/E3; and a light chainfrom subtype BoNT/F1, BoNT/F2, BoNT/F3 and BoNT/F4. A Clostridial toxinlight chain subtype can function in substantially the same manner as thereference Clostridial toxin light chain on which the Clostridial toxinlight chain subtype is based, and can be substituted for the referenceClostridial toxin light chain in any aspect of the present invention.

As used herein, the term “non-naturally occurring Clostridial toxinlight chain variant” means any Clostridial toxin light chain producedwith the aid of human manipulation, including, without limitation,Clostridial toxin light chains produced by genetic engineering usingrandom mutagenesis or rational design and Clostridial toxin light chainsproduced by chemical synthesis. Non-limiting examples of non-naturallyoccurring Clostridial toxin light chain variants include, e.g.,conservative Clostridial toxin light chain variants, non-conservativeClostridial toxin light chain variants, Clostridial toxin light chainchimeric variants and active Clostridial toxin light chain fragments.

As used herein, the term “conservative Clostridial toxin light chainvariant” means a Clostridial toxin light chain that has at least oneamino acid substituted by another amino acid or an amino acid analogthat has at least one property similar to that of the original aminoacid from the reference Clostridial toxin light chain sequence (Table1). Examples of properties include, without limitation, similar size,topography, charge, hydrophobicity, hydrophilicity, lipophilicity,covalent-bonding capacity, hydrogen-bonding capacity, a physicochemicalproperty, of the like, or any combination thereof. A conservativeClostridial toxin light chain variant can function in substantially thesame manner as the reference Clostridial toxin light chain on which theconservative Clostridial toxin light chain variant is based, and can besubstituted for the reference Clostridial toxin light chain in anyaspect of the present invention. A conservative Clostridial toxin lightchain variant may substitute one or more amino acids, two or more aminoacids, three or more amino acids, four or more amino acids, five or moreamino acids, ten or more amino acids, 20 or more amino acids, 30 or moreamino acids, 40 or more amino acids, 50 or more amino acids, 100 or moreamino acids, 200 or more amino acids, 300 or more amino acids, 400 ormore amino acids, or 500 or more amino acids from the referenceClostridial toxin light chain on which the conservative Clostridialtoxin light chain variant is based. A conservative Clostridial toxinlight chain variant can also substitute at least 10 contiguous aminoacids, at least 15 contiguous amino acids, at least 20 contiguous aminoacids, or at least 25 contiguous amino acids from the referenceClostridial toxin light chain on which the conservative Clostridialtoxin light chain variant is based, that possess at least 50% amino acididentity, 65% amino acid identity, 75% amino acid identity, 85% aminoacid identity or 95% amino acid identity to the reference Clostridialtoxin light chain on which the conservative Clostridial toxin lightchain variant is based. Non-limiting examples of a conservativeClostridial toxin light chain variant include, e.g., conservative BoNT/Alight chain variants, conservative BoNT/B light chain variants,conservative BoNT/C1 light chain variants, conservative BoNT/D lightchain variants, conservative BoNT/E light chain variants, conservativeBoNT/F light chain variants, conservative BoNT/G light chain variants,and conservative TeNT light chain variants.

As used herein, the term “non-conservative Clostridial toxin light chainvariant” means a Clostridial toxin light chain in which 1) at least oneamino acid is deleted from the reference Clostridial toxin light chainon which the non-conservative Clostridial toxin light chain variant isbased; 2) at least one amino acid added to the reference Clostridialtoxin light chain on which the non-conservative Clostridial toxin lightchain is based; or 3) at least one amino acid is substituted by anotheramino acid or an amino acid analog that does not share any propertysimilar to that of the original amino acid from the referenceClostridial toxin light chain sequence (Table 1). A non-conservativeClostridial toxin light chain variant can function in substantially thesame manner as the reference Clostridial toxin light chain on which thenon-conservative Clostridial toxin light chain variant is based, and canbe substituted for the reference Clostridial toxin light chain in anyaspect of the present invention. A non-conservative Clostridial toxinlight chain variant can delete one or more amino acids, two or moreamino acids, three or more amino acids, four or more amino acids, fiveor more amino acids, and ten or more amino acids from the referenceClostridial toxin light chain on which the non-conservative Clostridialtoxin light chain variant is based. A non-conservative Clostridial toxinlight chain variant can add one or more amino acids, two or more aminoacids, three or more amino acids, four or more amino acids, five or moreamino acids, and ten or more amino acids to the reference Clostridialtoxin light chain on which the non-conservative Clostridial toxin lightchain variant is based. A non-conservative Clostridial toxin light chainvariant may substitute one or more amino acids, two or more amino acids,three or more amino acids, four or more amino acids, five or more aminoacids, ten or more amino acids, 20 or more amino acids, 30 or more aminoacids, 40 or more amino acids, 50 or more amino acids, 100 or more aminoacids, 200 or more amino acids, 300 or more amino acids, 400 or moreamino acids, or 500 or more amino acids from the reference Clostridialtoxin light chain on which the non-conservative Clostridial toxin lightchain variant is based. A non-conservative Clostridial toxin light chainvariant can also substitute at least 10 contiguous amino acids, at least15 contiguous amino acids, at least 20 contiguous amino acids, or atleast 25 contiguous amino acids from the reference Clostridial toxinlight chain on which the non-conservative Clostridial toxin light chainvariant is based, that possess at least 50% amino acid identity, 65%amino acid identity, 75% amino acid identity, 85% amino acid identity or95% amino acid identity to the reference Clostridial toxin light chainon which the non-conservative Clostridial toxin light chain variant isbased. Non-limiting examples of a non-conservative Clostridial toxinlight chain variant include, e.g., non-conservative BoNT/A light chainvariants, non-conservative BoNT/B light chain variants, non-conservativeBoNT/C1 light chain variants, non-conservative BoNT/D light chainvariants, non-conservative BoNT/E light chain variants, non-conservativeBoNT/F light chain variants, non-conservative BoNT/G light chainvariants, and non-conservative TeNT light chain variants.

As used herein, the term “Clostridial toxin light chain chimeric” meansa polypeptide comprising at least a portion of a Clostridial toxin lightchain and at least a portion of at least one other polypeptide to form atoxin light chain with at least one property different from thereference Clostridial toxin light chains of Table 1, with the provisothat this Clostridial toxin light chain chimeric is still capable ofspecifically targeting the core components of the neurotransmitterrelease apparatus and thus participate in executing the overall cellularmechanism whereby a Clostridial toxin proteolytically cleaves asubstrate. Such Clostridial toxin light chain chimerics are describedin, e.g., Lance E. Steward et al., Leucine-based Motif and ClostridialToxins, U.S. Patent Publication 2003/0027752 (Feb. 6, 2003); Lance E.Steward et al., Clostridial Neurotoxin Compositions and ModifiedClostridial Neurotoxins, U.S. Patent Publication 2003/0219462 (Nov. 27,2003); and Lance E. Steward et al., Clostridial Neurotoxin Compositionsand Modified Clostridial Neurotoxins, U.S. Patent Publication2004/0220386 (Nov. 4, 2004).

As used herein, the term “active Clostridial toxin light chain fragment”means any of a variety of Clostridial toxin fragments comprising thelight chain can be useful in aspects of the present invention with theproviso that these light chain fragments can specifically target thecore components of the neurotransmitter release apparatus and thusparticipate in executing the overall cellular mechanism whereby aClostridial toxin proteolytically cleaves a substrate. The light chainsof Clostridial toxins are approximately 420-460 amino acids in lengthand comprise an enzymatic domain (Table 1). Research has shown that theentire length of a Clostridial toxin light chain is not necessary forthe enzymatic activity of the enzymatic domain. As a non-limitingexample, the first eight amino acids of the BoNT/A light chain (residues1-8 of SEQ ID NO: 1) are not required for enzymatic activity. As anothernon-limiting example, the first eight amino acids of the TeNT lightchain (residues 1-8 of SEQ ID NO: 8) are not required for enzymaticactivity. Likewise, the carboxyl-terminus of the light chain is notnecessary for activity. As a non-limiting example, the last 32 aminoacids of the BoNT/A light chain (residues 417-448 of SEQ ID NO: 1) arenot required for enzymatic activity. As another non-limiting example,the last 31 amino acids of the TeNT light chain (residues 427-457 of SEQID NO: 8) are not required for enzymatic activity. Thus, aspects of thisembodiment can include Clostridial toxin light chains comprising anenzymatic domain having a length of, e.g., at least 350 amino acids, atleast 375 amino acids, at least 400 amino acids, at least 425 aminoacids and at least 450 amino acids. Other aspects of this embodiment caninclude Clostridial toxin light chains comprising an enzymatic domainhaving a length of, e.g., at most 350 amino acids, at most 375 aminoacids, at most 400 amino acids, at most 425 amino acids and at most 450amino acids.

Any of a variety of sequence alignment methods can be used to determinepercent identity of naturally-occurring Clostridial toxin light chainvariants and non-naturally-occurring Clostridial toxin light chainvariants, including, without limitation, global methods, local methodsand hybrid methods, such as, e.g., segment approach methods. Protocolsto determine percent identity are routine procedures within the scope ofone skilled in the art and from the teaching herein.

Global methods align sequences from the beginning to the end of themolecule and determine the best alignment by adding up scores ofindividual residue pairs and by imposing gap penalties. Non-limitingmethods include, e.g., CLUSTAL W, see, e.g., Julie D. Thompson et al.,CLUSTAL W: Improving the Sensitivity of Progressive Multiple SequenceAlignment Through Sequence Weighting, Position-Specific Gap Penaltiesand Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680(1994); and iterative refinement, see, e.g., Osamu Gotoh, SignificantImprovement in Accuracy of Multiple Protein Sequence Alignments byIterative Refinement as Assessed by Reference to Structural Alignments,264(4) J. Mol. Biol. 823-838 (1996).

Local methods align sequences by identifying one or more conservedmotifs shared by all of the input sequences. Non-limiting methodsinclude, e.g., Match-box, see, e.g., Eric Depiereux and Ernest Feytmans,Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignmentof Several Protein Sequences, 8(5) CABIOS 501-509 (1992); Gibbssampling, see, e.g., C. E. Lawrence et al., Detecting Subtle SequenceSignals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131)Science 208-214 (1993); Align-M, see, e.g., Ivo Van Walle et al.,Align-M—A New Algorithm for Multiple Alignment of Highly DivergentSequences, 20(9) Bioinformatics: 1428-1435 (2004).

Hybrid methods combine functional aspects of both global and localalignment methods. Non-limiting methods include, e.g.,segment-to-segment comparison, see, e.g., Burkhard Morgenstern et al.,Multiple DNA and Protein Sequence Alignment Based On Segment-To-SegmentComparison, 93(22) Proc. Natl. Acad. Sci. U.S.A. 12098-12103 (1996);T-Coffee, see, e.g., Cédric Notredame et al., T-Coffee: A NovelAlgorithm for Multiple Sequence Alignment, 302(1) J. Mol. Biol. 205-217(2000); MUSCLE, see, e.g., Robert C. Edgar, MUSCLE: Multiple SequenceAlignment With High Score Accuracy and High Throughput, 32(5) NucleicAcids Res. 1792-1797 (2004); and DIALIGN-T, see, e.g., Amarendran RSubramanian et al., DIALIGN-T: An Improved Algorithm for Segment-BasedMultiple Sequence Alignment, 6(1) BMC Bioinformatics 66 (2005).

Thus, in an embodiment, a modified Clostridial toxin disclosed in thepresent specification comprises a Clostridial toxin enzymatic domain. Inan aspect of this embodiment, a Clostridial toxin enzymatic domaincomprises a naturally occurring Clostridial toxin light chain variant,such as, e.g., a Clostridial toxin light chain isoform or a Clostridialtoxin light chain subtype. In another aspect of this embodiment, aClostridial toxin enzymatic domain comprises a non-naturally occurringClostridial toxin light chain variant, such as, e.g., a conservativeClostridial toxin light chain variant, a non-conservative Clostridialtoxin light chain variant, a Clostridial toxin chimeric light chain, anactive Clostridial toxin light chain fragment, or any combinationthereof.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBoNT/A light chain. In an aspect of this embodiment, a BoNT/A lightchain comprises amino acids 1-448 of SEQ ID NO: 1. In another aspect ofthis embodiment, a BoNT/A light chain comprises a naturally occurringBoNT/A light chain variant, such as, e.g., a light chain from a BoNT/Aisoform or a light chain from a BoNT/A subtype.

In another aspect of this embodiment, a BoNT/A light chain comprisesamino acids 1-448 of a naturally occurring BoNT/A light chain variant ofSEQ ID NO: 1, such as, e.g., amino acids 1-448 of a BoNT/A isoform ofSEQ ID NO: 1 or amino acids 1-448 of a BoNT/A subtype of SEQ ID NO: 1.In still another aspect of this embodiment, a BoNT/A light chaincomprises a non-naturally occurring BoNT/A light chain variant, such as,e.g., a conservative BoNT/A light chain variant, a non-conservativeBoNT/A light chain variant, a BoNT/A chimeric light chain, an activeBoNT/A light chain fragment, or any combination thereof. In stillanother aspect of this embodiment, a BoNT/A light chain comprises aminoacids 1-448 of a non-naturally occurring BoNT/A light chain variant ofSEQ ID NO: 1, such as, e.g., amino acids 1-448 of a conservative BoNT/Alight chain variant of SEQ ID NO: 1, amino acids 1-448 of anon-conservative BoNT/A light chain variant of SEQ ID NO: 1, amino acids1-448 of an active BoNT/A light chain fragment of SEQ ID NO: 1, or anycombination thereof.

In other aspects of this embodiment, a BoNT/A light chain comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 1-448 of SEQ ID NO: 1, at least 75% amino acid identity with aminoacids 1-448 of SEQ ID NO: 1, at least 80% amino acid identity with aminoacids 1-448 of SEQ ID NO: 1, at least 85% amino acid identity with aminoacids 1-448 of SEQ ID NO: 1, at least 90% amino acid identity with aminoacids 1-448 of SEQ ID NO: 1 or at least 95% amino acid identity withamino acids 1-448 of SEQ ID NO: 1. In yet other aspects of thisembodiment, a BoNT/A light chain comprises a polypeptide having, e.g.,at most 70% amino acid identity with amino acids 1-448 of SEQ ID NO: 1,at most 75% amino acid identity with amino acids 1-448 of SEQ ID NO: 1,at most 80% amino acid identity with amino acids 1-448 of SEQ ID NO: 1,at most 85% amino acid identity with amino acids 1-448 of SEQ ID NO: 1,at most 90% amino acid identity with amino acids 1-448 of SEQ ID NO: 1or at most 95% amino acid identity with amino acids 1-448 of SEQ ID NO:1.

In other aspects of this embodiment, a BoNT/A light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 1-448 of SEQ ID NO: 1. Inother aspects of this embodiment, a BoNT/A light chain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 1-448 of SEQ ID NO: 1. In yetother aspects of this embodiment, a BoNT/A light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-448 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/A light chain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletionsrelative to amino acids 1-448 of SEQ ID NO: 1. In still other aspects ofthis embodiment, a BoNT/A light chain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relativeto amino acids 1-448 of SEQ ID NO: 1. In other aspects of thisembodiment, a BoNT/A light chain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10, 20,30, 40, 50, 100 or 200 non-contiguous amino acid additions relative toamino acids 1-448 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-448 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/A light chain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutionsrelative to amino acids 1-448 of SEQ ID NO: 1. In yet other aspects ofthis embodiment, a BoNT/A light chain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative toamino acids 1-448 of SEQ ID NO: 1. In other aspects of this embodiment,a BoNT/A light chain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid deletions relative to amino acids 1-448 ofSEQ ID NO: 1. In still other aspects of this embodiment, a BoNT/A lightchain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid additions relative to amino acids 1-448 of SEQ IDNO: 1. In other aspects of this embodiment, a BoNT/A light chaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid additions relative to amino acids 1-448 of SEQ ID NO: 1.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBoNT/B light chain. In an aspect of this embodiment, a BoNT/B lightchain comprises amino acids 1-441 of SEQ ID NO: 2. In another aspect ofthis embodiment, a BoNT/B light chain comprises a naturally occurringBoNT/B light chain variant, such as, e.g., a light chain from a BoNT/Bisoform or a light chain from a BoNT/B subtype. In another aspect ofthis embodiment, a BoNT/B light chain comprises amino acids 1-441 of anaturally occurring BoNT/B light chain variant of SEQ ID NO: 2, such as,e.g., amino acids 1-441 of a BoNT/B isoform of SEQ ID NO: 2 or aminoacids 1-441 of a BoNT/B subtype of SEQ ID NO: 2. In still another aspectof this embodiment, a BoNT/B light chain comprises a non-naturallyoccurring BoNT/B light chain variant, such as, e.g., a conservativeBoNT/B light chain variant, a non-conservative BoNT/B light chainvariant, a BoNT/B chimeric light chain, an active BoNT/B light chainfragment, or any combination thereof. In still another aspect of thisembodiment, a BoNT/B light chain comprises amino acids 1-441 of anon-naturally occurring BoNT/B light chain variant of SEQ ID NO: 2, suchas, e.g., amino acids 1-441 of a conservative BoNT/B light chain variantof SEQ ID NO: 2, amino acids 1-441 of a non-conservative BoNT/B lightchain variant of SEQ ID NO: 2, amino acids 1-441 of an active BoNT/Blight chain fragment of SEQ ID NO: 2, or any combination thereof.

In other aspects of this embodiment, a BoNT/B light chain comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 1-441 of SEQ ID NO: 2, at least 75% amino acid identity with aminoacids 1-441 of SEQ ID NO: 2, at least 80% amino acid identity with aminoacids 1-441 of SEQ ID NO: 2, at least 85% amino acid identity with aminoacids 1-441 of SEQ ID NO: 2, at least 90% amino acid identity with aminoacids 1-441 of SEQ ID NO: 2 or at least 95% amino acid identity withamino acids 1-441 of SEQ ID NO: 2. In yet other aspects of thisembodiment, a BoNT/B light chain comprises a polypeptide having, e.g.,at most 70% amino acid identity with amino acids 1-441 of SEQ ID NO: 2,at most 75% amino acid identity with amino acids 1-441 of SEQ ID NO: 2,at most 80% amino acid identity with amino acids 1-441 of SEQ ID NO: 2,at most 85% amino acid identity with amino acids 1-441 of SEQ ID NO: 2,at most 90% amino acid identity with amino acids 1-441 of SEQ ID NO: 2or at most 95% amino acid identity with amino acids 1-441 of SEQ ID NO:2.

In other aspects of this embodiment, a BoNT/B light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 1-441 of SEQ ID NO: 2. Inother aspects of this embodiment, a BoNT/B light chain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 1-441 of SEQ ID NO: 2. In yetother aspects of this embodiment, a BoNT/B light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-441 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B light chain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletionsrelative to amino acids 1-441 of SEQ ID NO: 2. In still other aspects ofthis embodiment, a BoNT/B light chain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relativeto amino acids 1-441 of SEQ ID NO: 2. In other aspects of thisembodiment, a BoNT/B light chain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10, 20,30, 40, 50, 100 or 200 non-contiguous amino acid additions relative toamino acids 1-441 of SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-441 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B light chain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutionsrelative to amino acids 1-441 of SEQ ID NO: 2. In yet other aspects ofthis embodiment, a BoNT/B light chain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative toamino acids 1-441 of SEQ ID NO: 2. In other aspects of this embodiment,a BoNT/B light chain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid deletions relative to amino acids 1-441 ofSEQ ID NO: 2. In still other aspects of this embodiment, a BoNT/B lightchain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid additions relative to amino acids 1-441 of SEQ IDNO: 2. In other aspects of this embodiment, a BoNT/B light chaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid additions relative to amino acids 1-441 of SEQ ID NO: 2.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBoNT/C1 light chain. In an aspect of this embodiment, a BoNT/C1 lightchain comprises amino acids 1-449 of SEQ ID NO: 3. In another aspect ofthis embodiment, a BoNT/C1 light chain comprises a naturally occurringBoNT/C1 light chain variant, such as, e.g., a light chain from a BoNT/C1isoform or a light chain from a BoNT/C1 subtype. In another aspect ofthis embodiment, a BoNT/C1 light chain comprises amino acids 1-449 of anaturally occurring BoNT/C1 light chain variant of SEQ ID NO: 3, suchas, e.g., amino acids 1-449 of a BoNT/C1 isoform of SEQ ID NO: 3 oramino acids 1-449 of a BoNT/C1 subtype of SEQ ID NO: 3. In still anotheraspect of this embodiment, a BoNT/C1 light chain comprises anon-naturally occurring BoNT/C1 light chain variant, such as, e.g., aconservative BoNT/C1 light chain variant, a non-conservative BoNT/C1light chain variant, a BoNT/C1 chimeric light chain, an active BoNT/C1light chain fragment, or any combination thereof. In still anotheraspect of this embodiment, a BoNT/C1 light chain comprises amino acids1-449 of a non-naturally occurring BoNT/C1 light chain variant of SEQ IDNO: 3, such as, e.g., amino acids 1-449 of a conservative BoNT/C1 lightchain variant of SEQ ID NO: 3, amino acids 1-449 of a non-conservativeBoNT/C1 light chain variant of SEQ ID NO: 3, amino acids 1-449 of anactive BoNT/C1 light chain fragment of SEQ ID NO: 3, or any combinationthereof.

In other aspects of this embodiment, a BoNT/C1 light chain comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 1-449 of SEQ ID NO: 3, at least 75% amino acid identity with aminoacids 1-449 of SEQ ID NO: 3, at least 80% amino acid identity with aminoacids 1-449 of SEQ ID NO: 3, at least 85% amino acid identity with aminoacids 1-449 of SEQ ID NO: 3, at least 90% amino acid identity with aminoacids 1-449 of SEQ ID NO: 3 or at least 95% amino acid identity withamino acids 1-449 of SEQ ID NO: 3. In yet other aspects of thisembodiment, a BoNT/C1 light chain comprises a polypeptide having, e.g.,at most 70% amino acid identity with amino acids 1-449 of SEQ ID NO: 3,at most 75% amino acid identity with amino acids 1-449 of SEQ ID NO: 3,at most 80% amino acid identity with amino acids 1-449 of SEQ ID NO: 3,at most 85% amino acid identity with amino acids 1-449 of SEQ ID NO: 3,at most 90% amino acid identity with amino acids 1-449 of SEQ ID NO: 3or at most 95% amino acid identity with amino acids 1-449 of SEQ ID NO:3.

In other aspects of this embodiment, a BoNT/C1 light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 1-449 of SEQ ID NO: 3. Inother aspects of this embodiment, a BoNT/C1 light chain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 1-449 of SEQ ID NO: 3. In yetother aspects of this embodiment, a BoNT/C1 light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-449 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 light chain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-449 of SEQ ID NO: 3. In stillother aspects of this embodiment, a BoNT/C1 light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 1-449 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 light chain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 1-449 of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-449 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 light chain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-449 of SEQ ID NO: 3. In yetother aspects of this embodiment, a BoNT/C1 light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 1-449 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 light chain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 1-449 of SEQ ID NO: 3. In still otheraspects of this embodiment, a BoNT/C1 light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 1-449 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 light chain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 1-449 of SEQ ID NO: 3.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBoNT/D light chain. In an aspect of this embodiment, a BoNT/D lightchain comprises amino acids 1-445 of SEQ ID NO: 4. In another aspect ofthis embodiment, a BoNT/D light chain comprises a naturally occurringBoNT/D light chain variant, such as, e.g., a light chain from a BoNT/Disoform or a light chain from a BoNT/D subtype. In another aspect ofthis embodiment, a BoNT/D light chain comprises amino acids 1-445 of anaturally occurring BoNT/D light chain variant of SEQ ID NO: 4, such as,e.g., amino acids 1-445 of a BoNT/D isoform of SEQ ID NO: 4 or aminoacids 1-445 of a BoNT/D subtype of SEQ ID NO: 4. In still another aspectof this embodiment, a BoNT/D light chain comprises a non-naturallyoccurring BoNT/D light chain variant, such as, e.g., a conservativeBoNT/D light chain variant, a non-conservative BoNT/D light chainvariant, a BoNT/D chimeric light chain, an active BoNT/D light chainfragment, or any combination thereof. In still another aspect of thisembodiment, a BoNT/D light chain comprises amino acids 1-445 of anon-naturally occurring BoNT/D light chain variant of SEQ ID NO: 4, suchas, e.g., amino acids 1-445 of a conservative BoNT/D light chain variantof SEQ ID NO: 4, amino acids 1-445 of a non-conservative BoNT/D lightchain variant of SEQ ID NO: 4, amino acids 1-445 of an active BoNT/Dlight chain fragment of SEQ ID NO: 4, or any combination thereof.

In other aspects of this embodiment, a BoNT/D light chain comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 1-445 of SEQ ID NO: 4, at least 75% amino acid identity with aminoacids 1-445 of SEQ ID NO: 4, at least 80% amino acid identity with aminoacids 1-445 of SEQ ID NO: 4, at least 85% amino acid identity with aminoacids 1-445 of SEQ ID NO: 4, at least 90% amino acid identity with aminoacids 1-445 of SEQ ID NO: 4 or at least 95% amino acid identity withamino acids 1-445 of SEQ ID NO: 4. In yet other aspects of thisembodiment, a BoNT/D light chain comprises a polypeptide having, e.g.,at most 70% amino acid identity with amino acids 1-445 of SEQ ID NO: 4,at most 75% amino acid identity with amino acids 1-445 of SEQ ID NO: 4,at most 80% amino acid identity with amino acids 1-445 of SEQ ID NO: 4,at most 85% amino acid identity with amino acids 1-445 of SEQ ID NO: 4,at most 90% amino acid identity with amino acids 1-445 of SEQ ID NO: 4or at most 95% amino acid identity with amino acids 1-445 of SEQ ID NO:4.

In other aspects of this embodiment, a BoNT/D light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 1-445 of SEQ ID NO: 4. Inother aspects of this embodiment, a BoNT/D light chain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 1-445 of SEQ ID NO: 4. In yetother aspects of this embodiment, a BoNT/D light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-445 of SEQ ID NO: 4. In otheraspects of this embodiment, a BoNT/D light chain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletionsrelative to amino acids 1-445 of SEQ ID NO: 4. In still other aspects ofthis embodiment, a BoNT/D light chain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relativeto amino acids 1-445 of SEQ ID NO: 4. In other aspects of thisembodiment, a BoNT/D light chain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10, 20,30, 40, 50, 100 or 200 non-contiguous amino acid additions relative toamino acids 1-445 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-445 of SEQ ID NO: 4. In otheraspects of this embodiment, a BoNT/D light chain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutionsrelative to amino acids 1-445 of SEQ ID NO: 4. In yet other aspects ofthis embodiment, a BoNT/D light chain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative toamino acids 1-445 of SEQ ID NO: 4. In other aspects of this embodiment,a BoNT/D light chain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid deletions relative to amino acids 1-445 ofSEQ ID NO: 4. In still other aspects of this embodiment, a BoNT/D lightchain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid additions relative to amino acids 1-445 of SEQ IDNO: 4. In other aspects of this embodiment, a BoNT/D light chaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid additions relative to amino acids 1-445 of SEQ ID NO: 4.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBoNT/E light chain. In an aspect of this embodiment, a BoNT/E lightchain comprises amino acids 1-422 of SEQ ID NO: 5. In another aspect ofthis embodiment, a BoNT/E light chain comprises a naturally occurringBoNT/E light chain variant, such as, e.g., a light chain from a BoNT/Eisoform or a light chain from a BoNT/E subtype. In another aspect ofthis embodiment, a BoNT/E light chain comprises amino acids 1-422 of anaturally occurring BoNT/E light chain variant of SEQ ID NO: 5, such as,e.g., amino acids 1-422 of a BoNT/E isoform of SEQ ID NO: 5 or aminoacids 1-422 of a BoNT/E subtype of SEQ ID NO: 5. In still another aspectof this embodiment, a BoNT/E light chain comprises a non-naturallyoccurring BoNT/E light chain variant, such as, e.g., a conservativeBoNT/E light chain variant, a non-conservative BoNT/E light chainvariant, a BoNT/E chimeric light chain, an active BoNT/E light chainfragment, or any combination thereof. In still another aspect of thisembodiment, a BoNT/E light chain comprises amino acids 1-422 of anon-naturally occurring BoNT/E light chain variant of SEQ ID NO: 5, suchas, e.g., amino acids 1-422 of a conservative BoNT/E light chain variantof SEQ ID NO: 5, amino acids 1-422 of a non-conservative BoNT/E lightchain variant of SEQ ID NO: 5, amino acids 1-422 of an active BoNT/Elight chain fragment of SEQ ID NO: 5, or any combination thereof.

In other aspects of this embodiment, a BoNT/E light chain comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 1-422 of SEQ ID NO: 5, at least 75% amino acid identity with aminoacids 1-422 of SEQ ID NO: 5, at least 80% amino acid identity with aminoacids 1-422 of SEQ ID NO: 5, at least 85% amino acid identity with aminoacids 1-422 of SEQ ID NO: 5, at least 90% amino acid identity with aminoacids 1-422 of SEQ ID NO: 5 or at least 95% amino acid identity withamino acids 1-422 of SEQ ID NO: 5. In yet other aspects of thisembodiment, a BoNT/E light chain comprises a polypeptide having, e.g.,at most 70% amino acid identity with amino acids 1-422 of SEQ ID NO: 5,at most 75% amino acid identity with amino acids 1-422 of SEQ ID NO: 5,at most 80% amino acid identity with amino acids 1-422 of SEQ ID NO: 5,at most 85% amino acid identity with amino acids 1-422 of SEQ ID NO: 5,at most 90% amino acid identity with amino acids 1-422 of SEQ ID NO: 5or at most 95% amino acid identity with amino acids 1-422 of SEQ ID NO:5.

In other aspects of this embodiment, a BoNT/E light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 1-422 of SEQ ID NO: 5. Inother aspects of this embodiment, a BoNT/E light chain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 1-422 of SEQ ID NO: 5. In yetother aspects of this embodiment, a BoNT/E light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-422 of SEQ ID NO: 5. In otheraspects of this embodiment, a BoNT/E light chain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletionsrelative to amino acids 1-422 of SEQ ID NO: 5. In still other aspects ofthis embodiment, a BoNT/E light chain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relativeto amino acids 1-422 of SEQ ID NO: 5. In other aspects of thisembodiment, a BoNT/E light chain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10, 20,30, 40, 50, 100 or 200 non-contiguous amino acid additions relative toamino acids 1-422 of SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-422 of SEQ ID NO: 5. In otheraspects of this embodiment, a BoNT/E light chain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutionsrelative to amino acids 1-422 of SEQ ID NO: 5. In yet other aspects ofthis embodiment, a BoNT/E light chain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative toamino acids 1-422 of SEQ ID NO: 5. In other aspects of this embodiment,a BoNT/E light chain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid deletions relative to amino acids 1-422 ofSEQ ID NO: 5. In still other aspects of this embodiment, a BoNT/E lightchain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid additions relative to amino acids 1-422 of SEQ IDNO: 5. In other aspects of this embodiment, a BoNT/E light chaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid additions relative to amino acids 1-422 of SEQ ID NO: 5.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBoNT/F light chain. In an aspect of this embodiment, a BoNT/F lightchain comprises amino acids 1-439 of SEQ ID NO: 6. In another aspect ofthis embodiment, a BoNT/F light chain comprises a naturally occurringBoNT/F light chain variant, such as, e.g., a light chain from a BoNT/Fisoform or a light chain from a BoNT/F subtype. In another aspect ofthis embodiment, a BoNT/F light chain comprises amino acids 1-439 of anaturally occurring BoNT/F light chain variant of SEQ ID NO: 6, such as,e.g., amino acids 1-439 of a BoNT/F isoform of SEQ ID NO: 6 or aminoacids 1-439 of a BoNT/F subtype of SEQ ID NO: 6. In still another aspectof this embodiment, a BoNT/F light chain comprises a non-naturallyoccurring BoNT/F light chain variant, such as, e.g., a conservativeBoNT/F light chain variant, a non-conservative BoNT/F light chainvariant, a BoNT/F chimeric light chain, an active BoNT/F light chainfragment, or any combination thereof. In still another aspect of thisembodiment, a BoNT/F light chain comprises amino acids 1-439 of anon-naturally occurring BoNT/F light chain variant of SEQ ID NO: 6, suchas, e.g., amino acids 1-439 of a conservative BoNT/F light chain variantof SEQ ID NO: 6, amino acids 1-439 of a non-conservative BoNT/F lightchain variant of SEQ ID NO: 6, amino acids 1-439 of an active BoNT/Flight chain fragment of SEQ ID NO: 6, or any combination thereof.

In other aspects of this embodiment, a BoNT/F light chain comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 1-439 of SEQ ID NO: 6, at least 75% amino acid identity with aminoacids 1-439 of SEQ ID NO: 6, at least 80% amino acid identity with aminoacids 1-439 of SEQ ID NO: 6, at least 85% amino acid identity with aminoacids 1-439 of SEQ ID NO: 6, at least 90% amino acid identity with aminoacids 1-439 of SEQ ID NO: 6 or at least 95% amino acid identity withamino acids 1-439 of SEQ ID NO: 6. In yet other aspects of thisembodiment, a BoNT/F light chain comprises a polypeptide having, e.g.,at most 70% amino acid identity with amino acids 1-439 of SEQ ID NO: 6,at most 75% amino acid identity with amino acids 1-439 of SEQ ID NO: 6,at most 80% amino acid identity with amino acids 1-439 of SEQ ID NO: 6,at most 85% amino acid identity with amino acids 1-439 of SEQ ID NO: 6,at most 90% amino acid identity with amino acids 1-439 of SEQ ID NO: 6or at most 95% amino acid identity with amino acids 1-439 of SEQ ID NO:6.

In other aspects of this embodiment, a BoNT/F light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 1-439 of SEQ ID NO: 6. Inother aspects of this embodiment, a BoNT/F light chain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 1-439 of SEQ ID NO: 6. In yetother aspects of this embodiment, a BoNT/F light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-439 of SEQ ID NO: 6. In otheraspects of this embodiment, a BoNT/F light chain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletionsrelative to amino acids 1-439 of SEQ ID NO: 6. In still other aspects ofthis embodiment, a BoNT/F light chain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relativeto amino acids 1-439 of SEQ ID NO: 6. In other aspects of thisembodiment, a BoNT/F light chain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10, 20,30, 40, 50, 100 or 200 non-contiguous amino acid additions relative toamino acids 1-439 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-439 of SEQ ID NO: 6. In otheraspects of this embodiment, a BoNT/F light chain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutionsrelative to amino acids 1-439 of SEQ ID NO: 6. In yet other aspects ofthis embodiment, a BoNT/F light chain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative toamino acids 1-439 of SEQ ID NO: 6. In other aspects of this embodiment,a BoNT/F light chain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid deletions relative to amino acids 1-439 ofSEQ ID NO: 6. In still other aspects of this embodiment, a BoNT/F lightchain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid additions relative to amino acids 1-439 of SEQ IDNO: 6. In other aspects of this embodiment, a BoNT/F light chaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid additions relative to amino acids 1-439 of SEQ ID NO: 6.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBoNT/G light chain. In an aspect of this embodiment, a BoNT/G lightchain comprises amino acids 1-446 of SEQ ID NO: 7. In another aspect ofthis embodiment, a BoNT/G light chain comprises a naturally occurringBoNT/G light chain variant, such as, e.g., a light chain from a BoNT/Gisoform or a light chain from a BoNT/G subtype. In another aspect ofthis embodiment, a BoNT/G light chain comprises amino acids 1-446 of anaturally occurring BoNT/G light chain variant of SEQ ID NO: 7, such as,e.g., amino acids 1-446 of a BoNT/G isoform of SEQ ID NO: 7 or aminoacids 1-446 of a BoNT/G subtype of SEQ ID NO: 7. In still another aspectof this embodiment, a BoNT/G light chain comprises a non-naturallyoccurring BoNT/G light chain variant, such as, e.g., a conservativeBoNT/G light chain variant, a non-conservative BoNT/G light chainvariant, a BoNT/G chimeric light chain, an active BoNT/G light chainfragment, or any combination thereof. In still another aspect of thisembodiment, a BoNT/G light chain comprises amino acids 1-446 of anon-naturally occurring BoNT/G light chain variant of SEQ ID NO: 7, suchas, e.g., amino acids 1-446 of a conservative BoNT/G light chain variantof SEQ ID NO: 7, amino acids 1-446 of a non-conservative BoNT/G lightchain variant of SEQ ID NO: 7, amino acids 1-446 of an active BoNT/Glight chain fragment of SEQ ID NO: 7, or any combination thereof.

In other aspects of this embodiment, a BoNT/G light chain comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 1-446 of SEQ ID NO: 7, at least 75% amino acid identity with aminoacids 1-446 of SEQ ID NO: 7, at least 80% amino acid identity with aminoacids 1-446 of SEQ ID NO: 7, at least 85% amino acid identity with aminoacids 1-446 of SEQ ID NO: 7, at least 90% amino acid identity with aminoacids 1-446 of SEQ ID NO: 7 or at least 95% amino acid identity withamino acids 1-446 of SEQ ID NO: 7. In yet other aspects of thisembodiment, a BoNT/G light chain comprises a polypeptide having, e.g.,at most 70% amino acid identity with amino acids 1-446 of SEQ ID NO: 7,at most 75% amino acid identity with amino acids 1-446 of SEQ ID NO: 7,at most 80% amino acid identity with amino acids 1-446 of SEQ ID NO: 7,at most 85% amino acid identity with amino acids 1-446 of SEQ ID NO: 7,at most 90% amino acid identity with amino acids 1-446 of SEQ ID NO: 7or at most 95% amino acid identity with amino acids 1-446 of SEQ ID NO:7.

In other aspects of this embodiment, a BoNT/G light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 1-446 of SEQ ID NO: 7. Inother aspects of this embodiment, a BoNT/G light chain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 1-446 of SEQ ID NO: 7. In yetother aspects of this embodiment, a BoNT/G light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-446 of SEQ ID NO: 7. In otheraspects of this embodiment, a BoNT/G light chain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletionsrelative to amino acids 1-446 of SEQ ID NO: 7. In still other aspects ofthis embodiment, a BoNT/G light chain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relativeto amino acids 1-446 of SEQ ID NO: 7. In other aspects of thisembodiment, a BoNT/G light chain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10, 20,30, 40, 50, 100 or 200 non-contiguous amino acid additions relative toamino acids 1-446 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-446 of SEQ ID NO: 7. In otheraspects of this embodiment, a BoNT/G light chain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutionsrelative to amino acids 1-446 of SEQ ID NO: 7. In yet other aspects ofthis embodiment, a BoNT/G light chain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative toamino acids 1-446 of SEQ ID NO: 7. In other aspects of this embodiment,a BoNT/G light chain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid deletions relative to amino acids 1-446 ofSEQ ID NO: 7. In still other aspects of this embodiment, a BoNT/G lightchain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid additions relative to amino acids 1-446 of SEQ IDNO: 7. In other aspects of this embodiment, a BoNT/G light chaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid additions relative to amino acids 1-446 of SEQ ID NO: 7.

In another embodiment, a Clostridial toxin enzymatic domain comprises aTeNT light chain. In an aspect of this embodiment, a TeNT light chaincomprises amino acids 1-457 of SEQ ID NO: 8. In another aspect of thisembodiment, a TeNT light chain comprises a naturally occurring TeNTlight chain variant, such as, e.g., a light chain from a TeNT isoform ora light chain from a TeNT subtype. In another aspect of this embodiment,a TeNT light chain comprises amino acids 1-457 of a naturally occurringTeNT light chain variant of SEQ ID NO: 8, such as, e.g., amino acids1-457 of a TeNT isoform of SEQ ID NO: 8 or amino acids 1-457 of a TeNTsubtype of SEQ ID NO: 8. In still another aspect of this embodiment, aTeNT light chain comprises a non-naturally occurring TeNT light chainvariant, such as, e.g., a conservative TeNT light chain variant, anon-conservative TeNT light chain variant, a TeNT chimeric light chain,an active TeNT light chain fragment, or any combination thereof. Instill another aspect of this embodiment, a TeNT light chain comprisesamino acids 1-457 of a non-naturally occurring TeNT light chain variantof SEQ ID NO: 8, such as, e.g., amino acids 1-457 of a conservative TeNTlight chain variant of SEQ ID NO: 8, amino acids 1-457 of anon-conservative TeNT light chain variant of SEQ ID NO: 8, amino acids1-457 of an active TeNT light chain fragment of SEQ ID NO: 8, or anycombination thereof.

In other aspects of this embodiment, a TeNT light chain comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 1-457 of SEQ ID NO: 8, at least 75% amino acid identity with aminoacids 1-457 of SEQ ID NO: 8, at least 80% amino acid identity with aminoacids 1-457 of SEQ ID NO: 8, at least 85% amino acid identity with aminoacids 1-457 of SEQ ID NO: 8, at least 90% amino acid identity with aminoacids 1-457 of SEQ ID NO: 8 or at least 95% amino acid identity withamino acids 1-457 of SEQ ID NO: 8. In yet other aspects of thisembodiment, a TeNT light chain comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 1-457 of SEQ ID NO: 8, atmost 75% amino acid identity with amino acids 1-457 of SEQ ID NO: 8, atmost 80% amino acid identity with amino acids 1-457 of SEQ ID NO: 8, atmost 85% amino acid identity with amino acids 1-457 of SEQ ID NO: 8, atmost 90% amino acid identity with amino acids 1-457 of SEQ ID NO: 8 orat most 95% amino acid identity with amino acids 1-457 of SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 1-457 of SEQ ID NO: 8. Inother aspects of this embodiment, a TeNT light chain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 1-457 of SEQ ID NO: 8. In yetother aspects of this embodiment, a TeNT light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-457 of SEQ ID NO: 8. In otheraspects of this embodiment, a TeNT light chain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletionsrelative to amino acids 1-457 of SEQ ID NO: 8. In still other aspects ofthis embodiment, a TeNT light chain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relativeto amino acids 1-457 of SEQ ID NO: 8. In other aspects of thisembodiment, a TeNT light chain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 non-contiguous amino acid additions relative to aminoacids 1-457 of SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-457 of SEQ ID NO: 8. In otheraspects of this embodiment, a TeNT light chain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutionsrelative to amino acids 1-457 of SEQ ID NO: 8. In yet other aspects ofthis embodiment, a TeNT light chain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative toamino acids 1-457 of SEQ ID NO: 8. In other aspects of this embodiment,a TeNT light chain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid deletions relative to amino acids 1-457 ofSEQ ID NO: 8. In still other aspects of this embodiment, a TeNT lightchain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid additions relative to amino acids 1-457 of SEQ IDNO: 8. In other aspects of this embodiment, a TeNT light chain comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 1-457 of SEQ ID NO: 8.

Aspects of the present invention provide, in part, a Clostridial toxintranslocation domain. As used herein, the term “Clostridial toxintranslocation domain” means any Clostridial toxin polypeptide that canexecute the translocation step of the intoxication process that mediatesClostridial toxin light chain translocation. Thus, a Clostridial toxintranslocation domain facilitates the movement of a Clostridial toxinlight chain across a membrane and encompasses the movement of aClostridial toxin light chain through the membrane an intracellularvesicle into the cytoplasm of a cell. Non-limiting examples of aClostridial toxin translocation domain include, e.g., a Clostridialtoxin H_(N) region such as, e.g., a BoNT/A H_(N) region, a BoNT/B H_(N)region, a BoNT/C1H_(N) region, a BoNT/D H_(N) region, a BoNT/E H_(N)region, a BoNT/F H_(N) region, a BoNT/G H_(N) region, and a TeNT H_(N)region.

A Clostridial toxin translocation domain includes, without limitation,naturally occurring Clostridial toxin H_(N) region variants, such as,e.g., Clostridial toxin H_(N) region isoforms and Clostridial toxinH_(N) region subtypes; non-naturally occurring Clostridial toxin H_(N)region variants, such as, e.g., conservative Clostridial toxin H_(N)region variants, non-conservative Clostridial toxin H_(N) regionvariants, Clostridial toxin H_(N) region chimerics, active Clostridialtoxin H_(N) region fragments thereof, or any combination thereof.

As used herein, the term “Clostridial toxin H_(N) region variant,”whether naturally-occurring or non-naturally-occurring, means aClostridial toxin H_(N) region that has at least one amino acid changefrom the corresponding region of the disclosed reference sequences (seeTable 1) and can be described in percent identity to the correspondingregion of that reference sequence. Unless expressly indicated, allClostridial toxin H_(N) region variants disclosed in the presentspecification are capable of executing the translocation step of theintoxication process that mediates Clostridial toxin light chaintranslocation. As non-limiting examples, a BoNT/A H_(N) region variantcomprising amino acids 449-871 of SEQ ID NO: 1 will have at least oneamino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to the amino acid region 449-871 ofSEQ ID NO: 1; a BoNT/B H_(N) region variant comprising amino acids442-858 of SEQ ID NO: 2 will have at least one amino acid difference,such as, e.g., an amino acid substitution, deletion or addition, ascompared to the amino acid region 442-858 of SEQ ID NO: 2; aBoNT/C1H_(N) region variant comprising amino acids 450-866 of SEQ ID NO:3 will have at least one amino acid difference, such as, e.g., an aminoacid substitution, deletion or addition, as compared to the amino acidregion 450-866 of SEQ ID NO: 3; a BoNT/D H_(N) region variant comprisingamino acids 446-862 of SEQ ID NO: 4 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the amino acid region 446-862 of SEQ ID NO: 4;a BoNT/E H_(N) region variant comprising amino acids 423-845 of SEQ IDNO: 5 will have at least one amino acid difference, such as, e.g., anamino acid substitution, deletion or addition, as compared to the aminoacid region 423-845 of SEQ ID NO: 5; a BoNT/F H_(N) region variantcomprising amino acids 440-864 of SEQ ID NO: 6 will have at least oneamino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to the amino acid region 440-864 ofSEQ ID NO: 6; a BoNT/G H_(N) region variant comprising amino acids447-863 of SEQ ID NO: 7 will have at least one amino acid difference,such as, e.g., an amino acid substitution, deletion or addition, ascompared to the amino acid region 447-863 of SEQ ID NO: 7; and a TeNTH_(N) region variant comprising amino acids 458-879 of SEQ ID NO: 8 willhave at least one amino acid difference, such as, e.g., an amino acidsubstitution, deletion or addition, as compared to the amino acid region458-879 of SEQ ID NO: 8.

It is recognized by those of skill in the art that within each serotypeof Clostridial toxin there can be naturally occurring Clostridial toxinH_(N) region variants that differ somewhat in their amino acid sequence,and also in the nucleic acids encoding these proteins. For example,there are presently four BoNT/A subtypes, BoNT/A1, BoNT/A2, BoNT/A3 andBoNT/A4, with specific H_(N) region subtypes showing approximately 87%amino acid identity when compared to another BoNT/A H_(N) regionsubtype. As used herein, the term “naturally occurring Clostridial toxinH_(N) region variant” means any Clostridial toxin H_(N) region producedby a naturally-occurring process, including, without limitation,Clostridial toxin H_(N) region isoforms produced fromalternatively-spliced transcripts, Clostridial toxin H_(N) regionisoforms produced by spontaneous mutation and Clostridial toxin H_(N)region subtypes. A naturally occurring Clostridial toxin H_(N) regionvariant can function in substantially the same manner as the referenceClostridial toxin H_(N) region on which the naturally occurringClostridial toxin H_(N) region variant is based, and can be substitutedfor the reference Clostridial toxin H_(N) region in any aspect of thepresent invention. A naturally occurring Clostridial toxin H_(N) regionvariant may substitute one or more amino acids, two or more amino acids,three or more amino acids, four or more amino acids, five or more aminoacids, ten or more amino acids, 20 or more amino acids, 30 or more aminoacids, 40 or more amino acids, 50 or more amino acids or 100 or moreamino acids from the reference Clostridial toxin H_(N) region on whichthe naturally occurring Clostridial toxin H_(N) region variant is based.A naturally occurring Clostridial toxin H_(N) region variant can alsosubstitute at least 10 contiguous amino acids, at least 15 contiguousamino acids, at least 20 contiguous amino acids, or at least 25contiguous amino acids from the reference Clostridial toxin H_(N) regionon which the naturally occurring Clostridial toxin H_(N) region variantis based, that possess at least 50% amino acid identity, 65% amino acididentity, 75% amino acid identity, 85% amino acid identity or 95% aminoacid identity to the reference Clostridial toxin H_(N) region on whichthe naturally occurring Clostridial toxin H_(N) region variant is based.

A non-limiting example of a naturally occurring Clostridial toxin H_(N)region variant is a Clostridial toxin H_(N) region isoform such as,e.g., a BoNT/A H_(N) region isoform, a BoNT/B H_(N) region isoform, aBoNT/C1H_(N) region isoform, a BoNT/D H_(N) region isoform, a BoNT/EH_(N) region isoform, a BoNT/F H_(N) region isoform, a BoNT/G H_(N)region isoform, and a TeNT H_(N) region isoform. A Clostridial toxinH_(N) region isoform can function in substantially the same manner asthe reference Clostridial toxin H_(N) region on which the Clostridialtoxin H_(N) region isoform is based, and can be substituted for thereference Clostridial toxin H_(N) region in any aspect of the presentinvention.

Another non-limiting examples of a naturally occurring Clostridial toxinH_(N) region variant is a Clostridial toxin H_(N) region subtype suchas, e.g., a H_(N) region from subtype BoNT/A1, BoNT/A2, BoNT/A3 andBoNT/A4; a H_(N) region from subtype BoNT/B1, BoNT/B2, BoNT/B bivalentand BoNT/B nonproteolytic; a H_(N) region from subtype BoNT/C1-1 andBoNT/C1-2; a H_(N) region from subtype BoNT/E1, BoNT/E2 and BoNT/E3; anda H_(N) region from subtype BoNT/F1, BoNT/F2, BoNT/F3 and BoNT/F4. AClostridial toxin H_(N) region subtype can function in substantially thesame manner as the reference Clostridial toxin H_(N) region on which theClostridial toxin H_(N) region subtype is based, and can be substitutedfor the reference Clostridial toxin H_(N) region in any aspect of thepresent invention.

As used herein, the term “non-naturally occurring Clostridial toxinH_(N) region variant” means any Clostridial toxin H_(N) region producedwith the aid of human manipulation, including, without limitation,Clostridial toxin H_(N) regions produced by genetic engineering usingrandom mutagenesis or rational design and Clostridial toxin H_(N)regions produced by chemical synthesis. Non-limiting examples ofnon-naturally occurring Clostridial toxin H_(N) region variants include,e.g., conservative Clostridial toxin H_(N) region variants,non-conservative Clostridial toxin H_(N) region variants, Clostridialtoxin H_(N) region chimeric variants and active Clostridial toxin H_(N)region fragments.

As used herein, the term “conservative Clostridial toxin H_(N) regionvariant” means a Clostridial toxin H_(N) region that has at least oneamino acid substituted by another amino acid or an amino acid analogthat has at least one property similar to that of the original aminoacid from the reference Clostridial toxin H_(N) region sequence (Table1). Examples of properties include, without limitation, similar size,topography, charge, hydrophobicity, hydrophilicity, lipophilicity,covalent-bonding capacity, hydrogen-bonding capacity, a physicochemicalproperty, of the like, or any combination thereof. A conservativeClostridial toxin H_(N) region variant can function in substantially thesame manner as the reference Clostridial toxin H_(N) region on which theconservative Clostridial toxin H_(N) region variant is based, and can besubstituted for the reference Clostridial toxin H_(N) region in anyaspect of the present invention. A conservative Clostridial toxin H_(N)region variant may substitute one or more amino acids, two or more aminoacids, three or more amino acids, four or more amino acids, five or moreamino acids, ten or more amino acids, 20 or more amino acids, 30 or moreamino acids, 40 or more amino acids, 50 or more amino acids, 100 or moreamino acids, 200 or more amino acids, 300 or more amino acids, 400 ormore amino acids, or 500 or more amino acids from the referenceClostridial toxin H_(N) region on which the conservative Clostridialtoxin H_(N) region variant is based. A conservative Clostridial toxinH_(N) region variant can also substitute at least 10 contiguous aminoacids, at least 15 contiguous amino acids, at least 20 contiguous aminoacids, or at least 25 contiguous amino acids from the referenceClostridial toxin H_(N) region on which the conservative Clostridialtoxin H_(N) region variant is based, that possess at least 50% aminoacid identity, 65% amino acid identity, 75% amino acid identity, 85%amino acid identity or 95% amino acid identity to the referenceClostridial toxin H_(N) region on which the conservative Clostridialtoxin H_(N) region variant is based. Non-limiting examples of aconservative Clostridial toxin H_(N) region variant include, e.g.,conservative BoNT/A H_(N) region variants, conservative BoNT/B H_(N)region variants, conservative BoNT/C1H_(N) region variants, conservativeBoNT/D H_(N) region variants, conservative BoNT/E H_(N) region variants,conservative BoNT/F H_(N) region variants, conservative BoNT/G H_(N)region variants, and conservative TeNT H_(N) region variants.

As used herein, the term “non-conservative Clostridial toxin H_(N)region variant” means a Clostridial toxin H_(N) region in which 1) atleast one amino acid is deleted from the reference Clostridial toxinH_(N) region on which the non-conservative Clostridial toxin H_(N)region variant is based; 2) at least one amino acid added to thereference Clostridial toxin H_(N) region on which the non-conservativeClostridial toxin H_(N) region is based; or 3) at least one amino acidis substituted by another amino acid or an amino acid analog that doesnot share any property similar to that of the original amino acid fromthe reference Clostridial toxin H_(N) region sequence (Table 1). Anon-conservative Clostridial toxin H_(N) region variant can function insubstantially the same manner as the reference Clostridial toxin H_(N)region on which the non-conservative Clostridial toxin H_(N) regionvariant is based, and can be substituted for the reference Clostridialtoxin H_(N) region in any aspect of the present invention. Anon-conservative Clostridial toxin H_(N) region variant can delete oneor more amino acids, two or more amino acids, three or more amino acids,four or more amino acids, five or more amino acids, and ten or moreamino acids from the reference Clostridial toxin H_(N) region on whichthe non-conservative Clostridial toxin H_(N) region variant is based. Anon-conservative Clostridial toxin H_(N) region variant can add one ormore amino acids, two or more amino acids, three or more amino acids,four or more amino acids, five or more amino acids, and ten or moreamino acids to the reference Clostridial toxin H_(N) region on which thenon-conservative Clostridial toxin H_(N) region variant is based. Anon-conservative Clostridial toxin H_(N) region variant may substituteone or more amino acids, two or more amino acids, three or more aminoacids, four or more amino acids, five or more amino acids, ten or moreamino acids, 20 or more amino acids, 30 or more amino acids, 40 or moreamino acids, 50 or more amino acids, 100 or more amino acids, 200 ormore amino acids, 300 or more amino acids, 400 or more amino acids, or500 or more amino acids from the reference Clostridial toxin H_(N)region on which the non-conservative Clostridial toxin H_(N) regionvariant is based. A non-conservative Clostridial toxin H_(N) regionvariant can also substitute at least 10 contiguous amino acids, at least15 contiguous amino acids, at least 20 contiguous amino acids, or atleast 25 contiguous amino acids from the reference Clostridial toxinH_(N) region on which the non-conservative Clostridial toxin H_(N)region variant is based, that possess at least 50% amino acid identity,65% amino acid identity, 75% amino acid identity, 85% amino acididentity or 95% amino acid identity to the reference Clostridial toxinH_(N) region on which the non-conservative Clostridial toxin H_(N)region variant is based. Non-limiting examples of a non-conservativeClostridial toxin H_(N) region variant include, e.g., non-conservativeBoNT/A H_(N) region variants, non-conservative BoNT/B H_(N) regionvariants, non-conservative BoNT/C1H_(N) region variants,non-conservative BoNT/D H_(N) region variants, non-conservative BoNT/EH_(N) region variants, non-conservative BoNT/F H_(N) region variants,non-conservative BoNT/G H_(N) region variants, and non-conservative TeNTH_(N) region variants.

As used herein, the term “Clostridial toxin H_(N) region chimeric” meansa polypeptide comprising at least a portion of a Clostridial toxin H_(N)region and at least a portion of at least one other polypeptide to forma toxin H_(N) region with at least one property different from thereference Clostridial toxin H_(N) regions of Table 1, with the provisothat this Clostridial toxin H_(N) region chimeric is still capable ofspecifically targeting the core components of the neurotransmitterrelease apparatus and thus participate in executing the overall cellularmechanism whereby a Clostridial toxin proteolytically cleaves asubstrate.

As used herein, the term “active Clostridial toxin H_(N) regionfragment” means any of a variety of Clostridial toxin fragmentscomprising the H_(N) region can be useful in aspects of the presentinvention with the proviso that these active fragments can facilitatethe release of the LC from intracellular vesicles into the cytoplasm ofthe target cell and thus participate in executing the overall cellularmechanism whereby a Clostridial toxin proteolytically cleaves asubstrate. The H_(N) regions from the heavy chains of Clostridial toxinsare approximately 410-430 amino acids in length and comprise atranslocation domain (Table 1). Research has shown that the entirelength of a H_(N) region from a Clostridial toxin heavy chain is notnecessary for the translocating activity of the translocation domain.Thus, aspects of this embodiment can include Clostridial toxin H_(N)regions comprising a translocation domain having a length of, e.g., atleast 350 amino acids, at least 375 amino acids, at least 400 aminoacids and at least 425 amino acids. Other aspects of this embodiment caninclude Clostridial toxin H_(N) regions comprising translocation domainhaving a length of, e.g., at most 350 amino acids, at most 375 aminoacids, at most 400 amino acids and at most 425 amino acids.

Any of a variety of sequence alignment methods can be used to determinepercent identity of naturally-occurring Clostridial toxin H_(N) regionvariants and non-naturally-occurring Clostridial toxin H_(N) regionvariants, including, without limitation, global methods, local methodsand hybrid methods, such as, e.g., segment approach methods. Protocolsto determine percent identity are routine procedures within the scope ofone skilled in the art and from the teaching herein.

Thus, in an embodiment, a modified Clostridial toxin disclosed in thepresent specification comprises a Clostridial toxin translocationdomain. In an aspect of this embodiment, a Clostridial toxintranslocation domain comprises a naturally occurring Clostridial toxinH_(N) region variant, such as, e.g., a Clostridial toxin H_(N) regionisoform or a Clostridial toxin H_(N) region subtype. In another aspectof this embodiment, a Clostridial toxin translocation domain comprises anon-naturally occurring Clostridial toxin H_(N) region variant, such as,e.g., a conservative Clostridial toxin H_(N) region variant, anon-conservative Clostridial toxin H_(N) region variant, a Clostridialtoxin chimeric H_(N) region, an active Clostridial toxin H_(N) regionfragment, or any combination thereof.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/A H_(N) region. In an aspect of this embodiment, aBoNT/A H_(N) region comprises amino acids 449-871 of SEQ ID NO: 1. Inanother aspect of this embodiment, a BoNT/A H_(N) region comprises anaturally occurring BoNT/A H_(N) region variant, such as, e.g., a H_(N)region from a BoNT/A isoform or a H_(N) region from a BoNT/A subtype. Inanother aspect of this embodiment, a BoNT/A H_(N) region comprises aminoacids 449-871 of a naturally occurring BoNT/A H_(N) region variant ofSEQ ID NO: 1, such as, e.g., amino acids 449-871 of a BoNT/A isoform ofSEQ ID NO: 1 or amino acids 449-871 of a BoNT/A subtype of SEQ ID NO: 1.In still another aspect of this embodiment, a BoNT/A H_(N) regioncomprises a non-naturally occurring BoNT/A H_(N) region variant, suchas, e.g., a conservative BoNT/A H_(N) region variant, a non-conservativeBoNT/A H_(N) region variant, a BoNT/A chimeric H_(N) region, an activeBoNT/A H_(N) region fragment, or any combination thereof. In stillanother aspect of this embodiment, a BoNT/A H_(N) region comprises aminoacids 449-871 of a non-naturally occurring BoNT/A H_(N) region variantof SEQ ID NO: 1, such as, e.g., amino acids 449-871 of a conservativeBoNT/A H_(N) region variant of SEQ ID NO: 1, amino acids 449-871 of anon-conservative BoNT/A H_(N) region variant of SEQ ID NO: 1, aminoacids 449-871 of an active BoNT/A H_(N) region fragment of SEQ ID NO: 1,or any combination thereof.

In other aspects of this embodiment, a BoNT/A H_(N) region comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 449-871 of SEQ ID NO: 1, at least 75% amino acid identity withamino acids 449-871 of SEQ ID NO: 1, at least 80% amino acid identitywith amino acids 449-871 of SEQ ID NO: 1, at least 85% amino acididentity with amino acids 449-871 of SEQ ID NO: 1, at least 90% aminoacid identity with amino acids 449-871 of SEQ ID NO: 1 or at least 95%amino acid identity with amino acids 449-871 of SEQ ID NO: 1. In yetother aspects of this embodiment, a BoNT/A H_(N) region comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 449-871 of SEQ ID NO: 1, at most 75% amino acid identity withamino acids 449-871 of SEQ ID NO: 1, at most 80% amino acid identitywith amino acids 449-871 of SEQ ID NO: 1, at most 85% amino acididentity with amino acids 449-871 of SEQ ID NO: 1, at most 90% aminoacid identity with amino acids 449-871 of SEQ ID NO: 1 or at most 95%amino acid identity with amino acids 449-871 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 449-871 of SEQ ID NO: 1. Inother aspects of this embodiment, a BoNT/A H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 449-871 of SEQ ID NO: 1. Inyet other aspects of this embodiment, a BoNT/A H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 449-871 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/A H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 449-871 of SEQ ID NO: 1. In stillother aspects of this embodiment, a BoNT/A H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 449-871 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/A H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 449-871 of SEQ ID NO: In otheraspects of this embodiment, a BoNT/A H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 449-871 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/A H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 449-871 of SEQ ID NO: 1. In yetother aspects of this embodiment, a BoNT/A H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 449-871 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/A H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 449-871 of SEQ ID NO: 1. In stillother aspects of this embodiment, a BoNT/A H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 449-871 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/A H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 449-871 of SEQ ID NO: 1.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/B H_(N) region. In an aspect of this embodiment, aBoNT/B H_(N) region comprises amino acids 442-858 of SEQ ID NO: 2. Inanother aspect of this embodiment, a BoNT/B H_(N) region comprises anaturally occurring BoNT/B H_(N) region variant, such as, e.g., a H_(N)region from a BoNT/B isoform or a H_(N) region from a BoNT/B subtype. Inanother aspect of this embodiment, a BoNT/B H_(N) region comprises aminoacids 442-858 of a naturally occurring BoNT/B H_(N) region variant ofSEQ ID NO: 2, such as, e.g., amino acids 442-858 of a BoNT/B isoform ofSEQ ID NO: 2 or amino acids 442-858 of a BoNT/B subtype of SEQ ID NO: 2.In still another aspect of this embodiment, a BoNT/B H_(N) regioncomprises a non-naturally occurring BoNT/B H_(N) region variant, suchas, e.g., a conservative BoNT/B H_(N) region variant, a non-conservativeBoNT/B H_(N) region variant, a BoNT/B chimeric H_(N) region, an activeBoNT/B H_(N) region fragment, or any combination thereof. In stillanother aspect of this embodiment, a BoNT/B H_(N) region comprises aminoacids 442-858 of a non-naturally occurring BoNT/B H_(N) region variantof SEQ ID NO: 2, such as, e.g., amino acids 442-858 of a conservativeBoNT/B H_(N) region variant of SEQ ID NO: 2, amino acids 442-858 of anon-conservative BoNT/B H_(N) region variant of SEQ ID NO: 2, aminoacids 442-858 of an active BoNT/B H_(N) region fragment of SEQ ID NO: 2,or any combination thereof.

In other aspects of this embodiment, a BoNT/B H_(N) region comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 442-858 of SEQ ID NO: 2, at least 75% amino acid identity withamino acids 442-858 of SEQ ID NO: 2, at least 80% amino acid identitywith amino acids 442-858 of SEQ ID NO: 2, at least 85% amino acididentity with amino acids 442-858 of SEQ ID NO: 2, at least 90% aminoacid identity with amino acids 442-858 of SEQ ID NO: 2 or at least 95%amino acid identity with amino acids 442-858 of SEQ ID NO: 2. In yetother aspects of this embodiment, a BoNT/B H_(N) region comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 442-858 of SEQ ID NO: 2, at most 75% amino acid identity withamino acids 442-858 of SEQ ID NO: 2, at most 80% amino acid identitywith amino acids 442-858 of SEQ ID NO: 2, at most 85% amino acididentity with amino acids 442-858 of SEQ ID NO: 2, at most 90% aminoacid identity with amino acids 442-858 of SEQ ID NO: 2 or at most 95%amino acid identity with amino acids 442-858 of SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 442-858 of SEQ ID NO: 2. Inother aspects of this embodiment, a BoNT/B H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 442-858 of SEQ ID NO: 2. Inyet other aspects of this embodiment, a BoNT/B H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 442-858 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 442-858 of SEQ ID NO: 2. In stillother aspects of this embodiment, a BoNT/B H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 442-858 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 442-858 of SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 442-858 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 442-858 of SEQ ID NO: 2. In yetother aspects of this embodiment, a BoNT/B H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 442-858 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 442-858 of SEQ ID NO: 2. In stillother aspects of this embodiment, a BoNT/B H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 442-858 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 442-858 of SEQ ID NO: 2.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/C1H_(N) region. In an aspect of this embodiment, aBoNT/C1H_(N) region comprises amino acids 450-866 of SEQ ID NO: 3. Inanother aspect of this embodiment, a BoNT/C1H_(N) region comprises anaturally occurring BoNT/C1H_(N) region variant, such as, e.g., a H_(N)region from a BoNT/C1 isoform or a H_(N) region from a BoNT/C1 subtype.In another aspect of this embodiment, a BoNT/C1H_(N) region comprisesamino acids 450-866 of a naturally occurring BoNT/C1H_(N) region variantof SEQ ID NO: 3, such as, e.g., amino acids 450-866 of a BoNT/C1 isoformof SEQ ID NO: 3 or amino acids 450-866 of a BoNT/C1 subtype of SEQ IDNO: 3. In still another aspect of this embodiment, a BoNT/C1H_(N) regioncomprises a non-naturally occurring BoNT/C1H_(N) region variant, suchas, e.g., a conservative BoNT/C1H_(N) region variant, a non-conservativeBoNT/C1H_(N) region variant, a BoNT/C1 chimeric H_(N) region, an activeBoNT/C1H_(N) region fragment, or any combination thereof. In stillanother aspect of this embodiment, a BoNT/C1H_(N) region comprises aminoacids 450-866 of a non-naturally occurring BoNT/C1H_(N) region variantof SEQ ID NO: 3, such as, e.g., amino acids 450-866 of a conservativeBoNT/C1H_(N) region variant of SEQ ID NO: 3, amino acids 450-866 of anon-conservative BoNT/C1H_(N) region variant of SEQ ID NO: 3, aminoacids 450-866 of an active BoNT/C1H_(N) region fragment of SEQ ID NO: 3,or any combination thereof.

In other aspects of this embodiment, a BoNT/C1H_(N) region comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 450-866 of SEQ ID NO: 3, at least 75% amino acid identity withamino acids 450-866 of SEQ ID NO: 3, at least 80% amino acid identitywith amino acids 450-866 of SEQ ID NO: 3, at least 85% amino acididentity with amino acids 450-866 of SEQ ID NO: 3, at least 90% aminoacid identity with amino acids 450-866 of SEQ ID NO: 3 or at least 95%amino acid identity with amino acids 450-866 of SEQ ID NO: 3. In yetother aspects of this embodiment, a BoNT/C1H_(N) region comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 450-866 of SEQ ID NO: 3, at most 75% amino acid identity withamino acids 450-866 of SEQ ID NO: 3, at most 80% amino acid identitywith amino acids 450-866 of SEQ ID NO: 3, at most 85% amino acididentity with amino acids 450-866 of SEQ ID NO: 3, at most 90% aminoacid identity with amino acids 450-866 of SEQ ID NO: 3 or at most 95%amino acid identity with amino acids 450-866 of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 450-866 of SEQ ID NO: 3. Inother aspects of this embodiment, a BoNT/C1H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 450-866 of SEQ ID NO: 3. Inyet other aspects of this embodiment, a BoNT/C1H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 450-866 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 450-866 of SEQ ID NO: 3. In stillother aspects of this embodiment, a BoNT/C1H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 450-866 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 450-866 of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 450-866 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 450-866 of SEQ ID NO: 3. In yetother aspects of this embodiment, a BoNT/C1H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 450-866 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 450-866 of SEQ ID NO: 3. In stillother aspects of this embodiment, a BoNT/C1H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 450-866 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 450-866 of SEQ ID NO: 3.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/D H_(N) region. In an aspect of this embodiment, aBoNT/D H_(N) region comprises amino acids 446-862 of SEQ ID NO: 4. Inanother aspect of this embodiment, a BoNT/D H_(N) region comprises anaturally occurring BoNT/D H_(N) region variant, such as, e.g., a H_(N)region from a BoNT/D isoform or a H_(N) region from a BoNT/D subtype. Inanother aspect of this embodiment, a BoNT/D H_(N) region comprises aminoacids 446-862 of a naturally occurring BoNT/D H_(N) region variant ofSEQ ID NO: 4, such as, e.g., amino acids 446-862 of a BoNT/D isoform ofSEQ ID NO: 4 or amino acids 446-862 of a BoNT/D subtype of SEQ ID NO: 4.In still another aspect of this embodiment, a BoNT/D H_(N) regioncomprises a non-naturally occurring BoNT/D H_(N) region variant, suchas, e.g., a conservative BoNT/D H_(N) region variant, a non-conservativeBoNT/D H_(N) region variant, a BoNT/D chimeric H_(N) region, an activeBoNT/D H_(N) region fragment, or any combination thereof. In stillanother aspect of this embodiment, a BoNT/D H_(N) region comprises aminoacids 446-862 of a non-naturally occurring BoNT/D H_(N) region variantof SEQ ID NO: 4, such as, e.g., amino acids 446-862 of a conservativeBoNT/D H_(N) region variant of SEQ ID NO: 4, amino acids 446-862 of anon-conservative BoNT/D H_(N) region variant of SEQ ID NO: 4, aminoacids 446-862 of an active BoNT/D H_(N) region fragment of SEQ ID NO: 4,or any combination thereof.

In other aspects of this embodiment, a BoNT/D H_(N) region comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 446-862 of SEQ ID NO: 4, at least 75% amino acid identity withamino acids 446-862 of SEQ ID NO: 4, at least 80% amino acid identitywith amino acids 446-862 of SEQ ID NO: 4, at least 85% amino acididentity with amino acids 446-862 of SEQ ID NO: 4, at least 90% aminoacid identity with amino acids 446-862 of SEQ ID NO: 4 or at least 95%amino acid identity with amino acids 446-862 of SEQ ID NO: 4. In yetother aspects of this embodiment, a BoNT/D H_(N) region comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 446-862 of SEQ ID NO: 4, at most 75% amino acid identity withamino acids 446-862 of SEQ ID NO: 4, at most 80% amino acid identitywith amino acids 446-862 of SEQ ID NO: 4, at most 85% amino acididentity with amino acids 446-862 of SEQ ID NO: 4, at most 90% aminoacid identity with amino acids 446-862 of SEQ ID NO: 4 or at most 95%amino acid identity with amino acids 446-862 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 446-862 of SEQ ID NO: 4. Inother aspects of this embodiment, a BoNT/D H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 446-862 of SEQ ID NO: 4. Inyet other aspects of this embodiment, a BoNT/D H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 446-862 of SEQ ID NO: 4. In otheraspects of this embodiment, a BoNT/D H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 446-862 of SEQ ID NO: 4. In stillother aspects of this embodiment, a BoNT/D H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 446-862 of SEQ ID NO: 4. In otheraspects of this embodiment, a BoNT/D H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 446-862 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 446-862 of SEQ ID NO: 4. In otheraspects of this embodiment, a BoNT/D H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 446-862 of SEQ ID NO: 4. In yetother aspects of this embodiment, a BoNT/D H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 446-862 of SEQ ID NO: 4. In otheraspects of this embodiment, a BoNT/D H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 446-862 of SEQ ID NO: 4. In stillother aspects of this embodiment, a BoNT/D H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 446-862 of SEQ ID NO: 4. In otheraspects of this embodiment, a BoNT/D H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 446-862 of SEQ ID NO: 4.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/E H_(N) region. In an aspect of this embodiment, aBoNT/E H_(N) region comprises amino acids 423-845 of SEQ ID NO: 5. Inanother aspect of this embodiment, a BoNT/E H_(N) region comprises anaturally occurring BoNT/E H_(N) region variant, such as, e.g., a H_(N)region from a BoNT/E isoform or a H_(N) region from a BoNT/E subtype. Inanother aspect of this embodiment, a BoNT/E H_(N) region comprises aminoacids 423-845 of a naturally occurring BoNT/E H_(N) region variant ofSEQ ID NO: 5, such as, e.g., amino acids 423-845 of a BoNT/E isoform ofSEQ ID NO: 5 or amino acids 423-845 of a BoNT/E subtype of SEQ ID NO: 5.In still another aspect of this embodiment, a BoNT/E H_(N) regioncomprises a non-naturally occurring BoNT/E H_(N) region variant, suchas, e.g., a conservative BoNT/E H_(N) region variant, a non-conservativeBoNT/E H_(N) region variant, a BoNT/E chimeric H_(N) region, an activeBoNT/E H_(N) region fragment, or any combination thereof. In stillanother aspect of this embodiment, a BoNT/E H_(N) region comprises aminoacids 423-845 of a non-naturally occurring BoNT/E H_(N) region variantof SEQ ID NO: 5, such as, e.g., amino acids 423-845 of a conservativeBoNT/E H_(N) region variant of SEQ ID NO: 5, amino acids 423-845 of anon-conservative BoNT/E H_(N) region variant of SEQ ID NO: 5, aminoacids 423-845 of an active BoNT/E H_(N) region fragment of SEQ ID NO: 5,or any combination thereof.

In other aspects of this embodiment, a BoNT/E H_(N) region comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 423-845 of SEQ ID NO: 5, at least 75% amino acid identity withamino acids 423-845 of SEQ ID NO: 5, at least 80% amino acid identitywith amino acids 423-845 of SEQ ID NO: 5, at least 85% amino acididentity with amino acids 423-845 of SEQ ID NO: 5, at least 90% aminoacid identity with amino acids 423-845 of SEQ ID NO: 5 or at least 95%amino acid identity with amino acids 423-845 of SEQ ID NO: 5. In yetother aspects of this embodiment, a BoNT/E H_(N) region comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 423-845 of SEQ ID NO: 5, at most 75% amino acid identity withamino acids 423-845 of SEQ ID NO: 5, at most 80% amino acid identitywith amino acids 423-845 of SEQ ID NO: 5, at most 85% amino acididentity with amino acids 423-845 of SEQ ID NO: 5, at most 90% aminoacid identity with amino acids 423-845 of SEQ ID NO: 5 or at most 95%amino acid identity with amino acids 423-845 of SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 423-845 of SEQ ID NO: 5. Inother aspects of this embodiment, a BoNT/E H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 423-845 of SEQ ID NO: 5. Inyet other aspects of this embodiment, a BoNT/E H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 423-845 of SEQ ID NO: 5. In otheraspects of this embodiment, a BoNT/E H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 423-845 of SEQ ID NO: 5. In stillother aspects of this embodiment, a BoNT/E H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 423-845 of SEQ ID NO: 5. In otheraspects of this embodiment, a BoNT/E H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 423-845 of SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 423-845 of SEQ ID NO: 5. In otheraspects of this embodiment, a BoNT/E H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 423-845 of SEQ ID NO: 5. In yetother aspects of this embodiment, a BoNT/E H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 423-845 of SEQ ID NO: 5. In otheraspects of this embodiment, a BoNT/E H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 423-845 of SEQ ID NO: 5. In stillother aspects of this embodiment, a BoNT/E H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 423-845 of SEQ ID NO: 5. In otheraspects of this embodiment, a BoNT/E H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 423-845 of SEQ ID NO: 5.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/F H_(N) region. In an aspect of this embodiment, aBoNT/F H_(N) region comprises amino acids 440-864 of SEQ ID NO: 6. Inanother aspect of this embodiment, a BoNT/F H_(N) region comprises anaturally occurring BoNT/F H_(N) region variant, such as, e.g., a H_(N)region from a BoNT/F isoform or a H_(N) region from a BoNT/F subtype. Inanother aspect of this embodiment, a BoNT/F H_(N) region comprises aminoacids 440-864 of a naturally occurring BoNT/F H_(N) region variant ofSEQ ID NO: 6, such as, e.g., amino acids 440-864 of a BoNT/F isoform ofSEQ ID NO: 6 or amino acids 440-864 of a BoNT/F subtype of SEQ ID NO: 6.In still another aspect of this embodiment, a BoNT/F H_(N) regioncomprises a non-naturally occurring BoNT/F H_(N) region variant, suchas, e.g., a conservative BoNT/F H_(N) region variant, a non-conservativeBoNT/F H_(N) region variant, a BoNT/F chimeric H_(N) region, an activeBoNT/F H_(N) region fragment, or any combination thereof. In stillanother aspect of this embodiment, a BoNT/F H_(N) region comprises aminoacids 440-864 of a non-naturally occurring BoNT/F H_(N) region variantof SEQ ID NO: 6, such as, e.g., amino acids 440-864 of a conservativeBoNT/F H_(N) region variant of SEQ ID NO: 6, amino acids 440-864 of anon-conservative BoNT/F H_(N) region variant of SEQ ID NO: 6, aminoacids 440-864 of an active BoNT/F H_(N) region fragment of SEQ ID NO: 6,or any combination thereof.

In other aspects of this embodiment, a BoNT/F H_(N) region comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 440-864 of SEQ ID NO: 6, at least 75% amino acid identity withamino acids 440-864 of SEQ ID NO: 6, at least 80% amino acid identitywith amino acids 440-864 of SEQ ID NO: 6, at least 85% amino acididentity with amino acids 440-864 of SEQ ID NO: 6, at least 90% aminoacid identity with amino acids 440-864 of SEQ ID NO: 6 or at least 95%amino acid identity with amino acids 440-864 of SEQ ID NO: 6. In yetother aspects of this embodiment, a BoNT/F H_(N) region comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 440-864 of SEQ ID NO: 6, at most 75% amino acid identity withamino acids 440-864 of SEQ ID NO: 6, at most 80% amino acid identitywith amino acids 440-864 of SEQ ID NO: 6, at most 85% amino acididentity with amino acids 440-864 of SEQ ID NO: 6, at most 90% aminoacid identity with amino acids 440-864 of SEQ ID NO: 6 or at most 95%amino acid identity with amino acids 440-864 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 440-864 of SEQ ID NO: 6. Inother aspects of this embodiment, a BoNT/F H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 440-864 of SEQ ID NO: 6. Inyet other aspects of this embodiment, a BoNT/F H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 440-864 of SEQ ID NO: 6. In otheraspects of this embodiment, a BoNT/F H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 440-864 of SEQ ID NO: 6. In stillother aspects of this embodiment, a BoNT/F H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 440-864 of SEQ ID NO: 6. In otheraspects of this embodiment, a BoNT/F H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 440-864 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 440-864 of SEQ ID NO: 6. In otheraspects of this embodiment, a BoNT/F H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 440-864 of SEQ ID NO: 6. In yetother aspects of this embodiment, a BoNT/F H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 440-864 of SEQ ID NO: 6. In otheraspects of this embodiment, a BoNT/F H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 440-864 of SEQ ID NO: 6. In stillother aspects of this embodiment, a BoNT/F H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 440-864 of SEQ ID NO: 6. In otheraspects of this embodiment, a BoNT/F H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 440-864 of SEQ ID NO: 6.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/G H_(N) region. In an aspect of this embodiment, aBoNT/G H_(N) region comprises amino acids 447-863 of SEQ ID NO: 7. Inanother aspect of this embodiment, a BoNT/G H_(N) region comprises anaturally occurring BoNT/G H_(N) region variant, such as, e.g., a H_(N)region from a BoNT/G isoform or a H_(N) region from a BoNT/G subtype. Inanother aspect of this embodiment, a BoNT/G H_(N) region comprises aminoacids 447-863 of a naturally occurring BoNT/G H_(N) region variant ofSEQ ID NO: 7, such as, e.g., amino acids 447-863 of a BoNT/G isoform ofSEQ ID NO: 7 or amino acids 447-863 of a BoNT/G subtype of SEQ ID NO: 7.In still another aspect of this embodiment, a BoNT/G H_(N) regioncomprises a non-naturally occurring BoNT/G H_(N) region variant, suchas, e.g., a conservative BoNT/G H_(N) region variant, a non-conservativeBoNT/G H_(N) region variant, a BoNT/G chimeric H_(N) region, an activeBoNT/G H_(N) region fragment, or any combination thereof. In stillanother aspect of this embodiment, a BoNT/G H_(N) region comprises aminoacids 447-863 of a non-naturally occurring BoNT/G H_(N) region variantof SEQ ID NO: 7, such as, e.g., amino acids 447-863 of a conservativeBoNT/G H_(N) region variant of SEQ ID NO: 7, amino acids 447-863 of anon-conservative BoNT/G H_(N) region variant of SEQ ID NO: 7, aminoacids 447-863 of an active BoNT/G H_(N) region fragment of SEQ ID NO: 7,or any combination thereof.

In other aspects of this embodiment, a BoNT/G H_(N) region comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 447-863 of SEQ ID NO: 7, at least 75% amino acid identity withamino acids 447-863 of SEQ ID NO: 7, at least 80% amino acid identitywith amino acids 447-863 of SEQ ID NO: 7, at least 85% amino acididentity with amino acids 447-863 of SEQ ID NO: 7, at least 90% aminoacid identity with amino acids 447-863 of SEQ ID NO: 7 or at least 95%amino acid identity with amino acids 447-863 of SEQ ID NO: 7. In yetother aspects of this embodiment, a BoNT/G H_(N) region comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 447-863 of SEQ ID NO: 7, at most 75% amino acid identity withamino acids 447-863 of SEQ ID NO: 7, at most 80% amino acid identitywith amino acids 447-863 of SEQ ID NO: 7, at most 85% amino acididentity with amino acids 447-863 of SEQ ID NO: 7, at most 90% aminoacid identity with amino acids 447-863 of SEQ ID NO: 7 or at most 95%amino acid identity with amino acids 447-863 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 447-863 of SEQ ID NO: 7. Inother aspects of this embodiment, a BoNT/G H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 447-863 of SEQ ID NO: 7. Inyet other aspects of this embodiment, a BoNT/G H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 447-863 of SEQ ID NO: 7. In otheraspects of this embodiment, a BoNT/G H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 447-863 of SEQ ID NO: 7. In stillother aspects of this embodiment, a BoNT/G H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 447-863 of SEQ ID NO: 7. In otheraspects of this embodiment, a BoNT/G H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 447-863 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 447-863 of SEQ ID NO: 7. In otheraspects of this embodiment, a BoNT/G H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 447-863 of SEQ ID NO: 7. In yetother aspects of this embodiment, a BoNT/G H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 447-863 of SEQ ID NO: 7. In otheraspects of this embodiment, a BoNT/G H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 447-863 of SEQ ID NO: 7. In stillother aspects of this embodiment, a BoNT/G H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 447-863 of SEQ ID NO: 7. In otheraspects of this embodiment, a BoNT/G H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 447-863 of SEQ ID NO: 7.

In another embodiment, a Clostridial toxin translocation domaincomprises a TeNT H_(N) region. In an aspect of this embodiment, a TeNTH_(N) region comprises amino acids 458-879 of SEQ ID NO: 8. In anotheraspect of this embodiment, a TeNT H_(N) region comprises a naturallyoccurring TeNT H_(N) region variant, such as, e.g., a H_(N) region froma TeNT isoform or a H_(N) region from a TeNT subtype. In another aspectof this embodiment, a TeNT H_(N) region comprises amino acids 458-879 ofa naturally occurring TeNT H_(N) region variant of SEQ ID NO: 8, suchas, e.g., amino acids 458-879 of a TeNT isoform of SEQ ID NO: 8 or aminoacids 458-879 of a TeNT subtype of SEQ ID NO: 8. In still another aspectof this embodiment, a TeNT H_(N) region comprises a non-naturallyoccurring TeNT H_(N) region variant, such as, e.g., a conservative TeNTH_(N) region variant, a non-conservative TeNT H_(N) region variant, aTeNT chimeric H_(N) region, an active TeNT H_(N) region fragment, or anycombination thereof. In still another aspect of this embodiment, a TeNTH_(N) region comprises amino acids 458-879 of a non-naturally occurringTeNT H_(N) region variant of SEQ ID NO: 8, such as, e.g., amino acids458-879 of a conservative TeNT H_(N) region variant of SEQ ID NO: 8,amino acids 458-879 of a non-conservative TeNT H_(N) region variant ofSEQ ID NO: 8, amino acids 458-879 of an active TeNT H_(N) regionfragment of SEQ ID NO: 8, or any combination thereof.

In other aspects of this embodiment, a TeNT H_(N) region comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 458-879 of SEQ ID NO: 8, at least 75% amino acid identity withamino acids 458-879 of SEQ ID NO: 8, at least 80% amino acid identitywith amino acids 458-879 of SEQ ID NO: 8, at least 85% amino acididentity with amino acids 458-879 of SEQ ID NO: 8, at least 90% aminoacid identity with amino acids 458-879 of SEQ ID NO: 8 or at least 95%amino acid identity with amino acids 458-879 of SEQ ID NO: 8. In yetother aspects of this embodiment, a TeNT H_(N) region comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 458-879 of SEQ ID NO: 8, at most 75% amino acid identity withamino acids 458-879 of SEQ ID NO: 8, at most 80% amino acid identitywith amino acids 458-879 of SEQ ID NO: 8, at most 85% amino acididentity with amino acids 458-879 of SEQ ID NO: 8, at most 90% aminoacid identity with amino acids 458-879 of SEQ ID NO: 8 or at most 95%amino acid identity with amino acids 458-879 of SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 458-879 of SEQ ID NO: 8. Inother aspects of this embodiment, a TeNT H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 458-879 of SEQ ID NO: 8. Inyet other aspects of this embodiment, a TeNT H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 458-879 of SEQ ID NO: 8. In otheraspects of this embodiment, a TeNT H_(N) region comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletionsrelative to amino acids 458-879 of SEQ ID NO: 8. In still other aspectsof this embodiment, a TeNT H_(N) region comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relativeto amino acids 458-879 of SEQ ID NO: 8. In other aspects of thisembodiment, a TeNT H_(N) region comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 non-contiguous amino acid additions relative to aminoacids 458-879 of SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 458-879 of SEQ ID NO: 8. In otheraspects of this embodiment, a TeNT H_(N) region comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutionsrelative to amino acids 458-879 of SEQ ID NO: 8. In yet other aspects ofthis embodiment, a TeNT H_(N) region comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative toamino acids 458-879 of SEQ ID NO: 8. In other aspects of thisembodiment, a TeNT H_(N) region comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 contiguous amino acid deletions relative to aminoacids 458-879 of SEQ ID NO: 8. In still other aspects of thisembodiment, a TeNT H_(N) region comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 contiguous amino acid additions relative to aminoacids 458-879 of SEQ ID NO: 8. In other aspects of this embodiment, aTeNT H_(N) region comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid additions relative to amino acids 458-879of SEQ ID NO: 8.

Aspects of the present invention provide, in part, an enhanced targetingdomain. As used herein, the term “enhanced targeting domain” means anypolypeptide that can selectively bind to a non-Clostridial toxinreceptor system present on a Clostridial toxin target cell and initiatethe overall internalization mechanism whereby a Clostridial toxinintoxicates a target cell. As used herein, the term “selectively” meanshaving a highly preferred activity or effect. As used herein, the term“selectively bind” means a molecule is able to bind its target receptorsystem under physiological conditions, or in vitro conditionssubstantially approximating physiological conditions, to a statisticallysignificantly greater degree relative to other, non-target receptorsystems. Thus, with reference to an enhanced targeting domain of thepresent specification, there is a discriminatory binding of the enhancedtargeting domain to an non-Clostridial toxin receptor system.

An enhanced targeting domain disclosed in the present specificationfacilitates the binding activity of the modified Clostridial toxinsdisclosed in the present specification to a non-Clostridial toxinreceptor system located at the surface of a Clostridial toxin targetcell. As used herein, the term “binding activity” means that onemolecule is directly or indirectly contacting another molecule via atleast one intermolecular or intramolecular force, including, withoutlimitation, a covalent bond, an ionic bond, a metallic bond, a hydrogenbond, a hydrophobic interaction, a van der Waals interaction, and thelike, or any combination thereof. “Bound” and “bind” are consideredterms for binding.

As used herein, the term “binding affinity” means how strong amolecule's binding activity is for a particular receptor system. Ingeneral, high binding affinity results from greater intermolecular forcebetween a binding domain and its receptor system while low bindingaffinity involves less intermolecular force between the ligand and itsreceptor. High binding affinity involves a longer residence time for thebinding domain at its receptor binding site than is the case for lowbinding affinity. As such, a molecule with a high binding affinity meansa lower concentration of that molecule is required to maximally occupythe binding sites of a receptor system and trigger a physiologicalresponse. Conversely, low binding affinity means a relatively highconcentration of a molecule is required before the receptor bindingsites of a receptor system is maximally occupied and the maximumphysiological response is achieved. Thus, modified Clostridial toxinswith increased binding activity due to high binding affinity will allowadministration of reduced doses of the toxin, thereby reducing orpreventing unwanted side-effects associated with toxin dispersal intonon-targeted areas.

As used herein, the term “binding specificity” means how specific amolecule's binding activity is one particular receptor system. Ingeneral, high binding specificity results in a more exclusiveinteraction with one particular receptor system or subgroup of receptorsystems while low binding specificity results in a more promiscuousinteraction with a larger group of receptor systems. As such, a moleculewith a high binding specificity means that molecule will occupy thebinding sites of a particular receptor system and trigger aphysiological response. Conversely, low binding specificity means amolecule will occupy the binding sites of a many receptor systems andtrigger a multitude of physiological responses. Thus, modifiedClostridial toxins with increased binding activity due to high bindingspecificity will only target non-Clostridial toxin receptors present ona subgroup of Clostridial toxin target cells, thereby reducing the sideeffects associated with the targeting of all Clostridial toxin targetcells.

As used herein, the term “Clostridial toxin target cell” means a cellthat is a naturally occurring cell that a naturally occurringClostridial toxin is capable of intoxicating, including, withoutlimitation, motor neurons; sensory neurons; autonomic neurons; such as,e.g., sympathetic neurons and parasympathetic neurons; non-petidergicneurons, such as, e.g., cholinergic neurons, adrenergic neurons,noradrenergic neurons, serotonergic neurons, GABAergic neurons; andpeptidergic neurons, such as, e.g., Substance P neurons, Calcitonin GeneRelated Peptide neurons, vasoactive intestinal peptide neurons,Neuropeptide Y neurons, cholecystokinin neurons.

It is envisioned that any and all enhanced targeting domains thatexhibits a binding activity for a non-Clostridial toxin receptor systempresent on a naturally-occurring Clostridial toxin target cell can beused to practice aspects of the present invention, including, withoutlimitation, polypeptides that selectively bind to a receptor systempresent on a presynaptic membrane and polypeptides that selectively bindto a receptor system present on a postsynaptic membrane. Polypeptidesthat appear to bind to a receptor system present on a presynapticmembrane, include, without limitation, Glucagon like hormones, such as,e.g., secretin, glucagon-like peptides, pituitary adenylate cyclaseactivating peptide (PACAP), glicentin, glicentin-related polypeptide(GRPP), oxyntomodulin (OXY), vasoactive intestinal peptides (VIPs),gastric inhibitory polypeptide (GIP), and calcitonin-relatedpeptidesvisceral gut peptides; neurohormones, such as, e.g.,corticotropin-releasing hormone (CCRH) and parathyroid hormone (PTH);neuroregulatory cytokines, such as, e.g., ciliary neurotrophic factor(CNTF), glycophorin-A (GPA), leukemia inhibitory factor (LIF),interleukins (ILs), onostatin M, cardiotrophin-1 (CT-1),cardiotrophin-like cytokine (CLC), neuroleukins, VEGF, insulin-likegrowth factors (IGFs), epidermal growth factor (EGF); neurotrophins,such as, e.g., nerve growth factors (NGFs), brain-derived growth factors(BDNFs), neurotrophin-3s (NT-3s) and neurotrophin-4/5s (NT-4/5s); growthfactors, such as, e.g., glial cell derived neurotrophic factor (GDNF),neurturin, persephrin, artemin, transformation growth factor betas(TGFβs), bone morphogenetic proteins (BMPs), growth and differentiationfactors (GDFs), activins; axon guidance signaling molecules, such as,e.g., netrins, semaphrorings and ephrins; sugar binding proteins, suchas, e.g., serum amyloid P, β-glucanase, sialidase, lectin, cryia,insecticidal delta-endotoxin, agglutinin, abrin and ricin; ligands thatselectively bind neurexins, such as, e.g., ligands for neurexin-1α andneurexin-1β; ligands for neurexin-2α and neurexin-2β; and ligands forneurexin-3α and neurexin-3β; and WNTs. Ligands that appear to bind to areceptor system present on a postsynaptic membrane, include, withoutlimitation, Ng-CAM(L1), NCAM, N-cadherin, Agrin-MUSK, basement membranepolypeptides, such as, e.g., laminin β-2.

An enhanced targeting domain includes, without limitation, naturallyoccurring enhanced targeting domain variants, such as, e.g., enhancedtargeting domain isoforms; non-naturally occurring enhanced targetingdomain variants, such as, e.g., conservative enhanced targeting domainvariants, non-conservative enhanced targeting domain variants, enhancedtargeting domain chimerics, active enhanced targeting domain fragmentsthereof, or any combination thereof.

As used herein, the term “variant,” when used to describe an enhancedtargeting domain variant, whether naturally-occurring ornon-naturally-occurring, means an enhanced targeting domain that has atleast one amino acid change from the corresponding region of thedisclosed reference sequences and can be described in percent identityto the corresponding region of that reference sequence. Unless expresslyindicated, all enhanced targeting domain variants disclosed in thepresent specification are capable of selectively binding to annon-Clostridial toxin receptor system present on a Clostridial toxintarget cell and initiate the overall internalization mechanism whereby aClostridial toxin intoxicates a target cell. As non-limiting examples, aglycogen-like peptide variant derived from amino acids 21-50 of SEQ IDNO: 9 will have at least one amino acid difference, such as, e.g., anamino acid substitution, deletion or addition, as compared to the aminoacid region 21-50 of SEQ ID NO: 9; a PACAP variant derived from aminoacids 132-158 of SEQ ID NO: 10 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the amino acid region 132-158 of SEQ ID NO: 10;a CCRH variant derived from amino acids 159-193 of SEQ ID NO: 19 willhave at least one amino acid difference, such as, e.g., an amino acidsubstitution, deletion or addition, as compared to the amino acid region159-193 of SEQ ID NO: 19; a PTH variant derived from amino acids 35-70of SEQ ID NO: 20 will have at least one amino acid difference, such as,e.g., an amino acid substitution, deletion or addition, as compared tothe amino acid region 35-70 of SEQ ID NO: 20; an IL-6 variant derivedfrom amino acids 57-210 of SEQ ID NO: 28 will have at least one aminoacid difference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the amino acid region 57-210 of SEQ ID NO: 28;a neuroleukin variant derived from amino acids 54-546 of SEQ ID NO: 31will have at least one amino acid difference, such as, e.g., an aminoacid substitution, deletion or addition, as compared to the amino acidregion 54-546 of SEQ ID NO: 31; an IGF-1 variant derived from aminoacids 52-109 of SEQ ID NO: 33 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the amino acid region 52-109 of SEQ ID NO: 33;and a NT-3 variant derived from amino acids 144-249 of SEQ ID NO: 38will have at least one amino acid difference, such as, e.g., an aminoacid substitution, deletion or addition, as compared to the amino acidregion 144-249 of SEQ ID NO: 38.

It is recognized by those of skill in the art that there can benaturally occurring enhanced targeting domain variants that differsomewhat in their amino acid sequence, and also in the nucleic acidsencoding these proteins. As used herein, the term “naturally occurringenhanced targeting domain variant” means any enhanced targeting domainproduced by a naturally-occurring process, including, withoutlimitation, enhanced targeting domain isoforms produced fromalternatively-spliced transcripts, enhanced targeting domain isoformsproduced by spontaneous mutation and enhanced targeting domain subtypes.A naturally occurring enhanced targeting domain variant can function insubstantially the same manner as the reference enhanced targeting domainon which the naturally occurring enhanced targeting domain variant isbased, and can be substituted for the reference enhanced targetingdomain in any aspect of the present invention. A naturally occurringenhanced targeting domain variant may substitute one or more aminoacids, two or more amino acids, three or more amino acids, four or moreamino acids, five or more amino acids, ten or more amino acids, 20 ormore amino acids, 30 or more amino acids, 40 or more amino acids, 50 ormore amino acids or 100 or more amino acids from the reference enhancedtargeting domain on which the naturally occurring enhanced targetingdomain variant is based. A naturally occurring enhanced targeting domainvariant can also substitute at least 10 contiguous amino acids, at least15 contiguous amino acids, at least 20 contiguous amino acids, or atleast 25 contiguous amino acids from the reference enhanced targetingdomain on which the naturally occurring enhanced targeting domainvariant is based, that possess at least 50% amino acid identity, 65%amino acid identity, 75% amino acid identity, 85% amino acid identity or95% amino acid identity to the reference enhanced targeting domain onwhich the naturally occurring enhanced targeting domain variant isbased.

A non-limiting example of a naturally occurring enhanced targetingdomain variant is an enhanced targeting domain isoform such as, e.g., aglucogen-like peptide isoform, a PACAP isoform, a CCRH isoform, a PTHisoform, an IL-6 isoform, a neuroleukin isoform, an IGF-1 isoform, and aNT-3 isoform. An enhanced targeting domain isoform can function insubstantially the same manner as the reference enhanced targeting domainon which the enhanced targeting domain isoform is based, and can besubstituted for the reference enhanced targeting domain in any aspect ofthe present invention.

As used herein, the term “non-naturally occurring enhanced targetingdomain variant” means any enhanced targeting domain produced with theaid of human manipulation, including, without limitation, enhancedtargeting domains produced by genetic engineering using randommutagenesis or rational design and enhanced targeting domains producedby chemical synthesis. Non-limiting examples of non-naturally occurringenhanced targeting domain variants include, e.g., conservative enhancedtargeting domain variants, non-conservative enhanced targeting domainvariants, enhanced targeting domain chimeric variants and activeenhanced targeting domain fragments.

As used herein, the term “conservative enhanced targeting domainvariant” means an enhanced targeting domain that has at least one aminoacid substituted by another amino acid or an amino acid analog that hasat least one property similar to that of the original amino acid fromthe reference enhanced targeting domain sequence. Examples of propertiesinclude, without limitation, similar size, topography, charge,hydrophobicity, hydrophilicity, lipophilicity, covalent-bondingcapacity, hydrogen-bonding capacity, a physicochemical property, of thelike, or any combination thereof. A conservative enhanced targetingdomain variant can function in substantially the same manner as thereference enhanced targeting domain on which the conservative enhancedtargeting domain variant is based, and can be substituted for thereference enhanced targeting domain in any aspect of the presentinvention. A conservative enhanced targeting domain variant maysubstitute one or more amino acids, two or more amino acids, three ormore amino acids, four or more amino acids, five or more amino acids,ten or more amino acids, 20 or more amino acids, 30 or more amino acids,40 or more amino acids, 50 or more amino acids, 100 or more amino acids,200 or more amino acids, 300 or more amino acids, 400 or more aminoacids, or 500 or more amino acids from the reference enhanced targetingdomain on which the conservative enhanced targeting domain variant isbased. A conservative enhanced targeting domain variant can alsosubstitute at least 10 contiguous amino acids, at least 15 contiguousamino acids, at least 20 contiguous amino acids, or at least 25contiguous amino acids from the reference enhanced targeting domain onwhich the conservative enhanced targeting domain variant is based, thatpossess at least 50% amino acid identity, 65% amino acid identity, 75%amino acid identity, 85% amino acid identity or 95% amino acid identityto the reference enhanced targeting domain on which the conservativeenhanced targeting domain variant is based. Non-limiting examples of aconservative enhanced targeting domain variant include, e.g.,conservative glucogen-like peptide variants, conservative PACAP regionvariants, conservative CCRH region variants, conservative PTH variants,conservative IL-6 variants, conservative neuroleukin variants,conservative IGF-1 variants, and conservative NT-3 region variants.

As used herein, the term “non-conservative enhanced targeting domainvariant” means an enhanced targeting domain in which 1) at least oneamino acid is deleted from the reference enhanced targeting domain onwhich the non-conservative enhanced targeting domain variant is based;2) at least one amino acid added to the reference enhanced targetingdomain on which the non-conservative enhanced targeting domain is based;or 3) at least one amino acid is substituted by another amino acid or anamino acid analog that does not share any property similar to that ofthe original amino acid from the reference enhanced targeting domainsequence. A non-conservative enhanced targeting domain variant canfunction in substantially the same manner as the reference enhancedtargeting domain on which the non-conservative enhanced targeting domainvariant is based, and can be substituted for the reference enhancedtargeting domain in any aspect of the present invention. Anon-conservative enhanced targeting domain variant can delete one ormore amino acids, two or more amino acids, three or more amino acids,four or more amino acids, five or more amino acids, and ten or moreamino acids from the reference enhanced targeting domain on which thenon-conservative enhanced targeting domain variant is based. Anon-conservative enhanced targeting domain variant can add one or moreamino acids, two or more amino acids, three or more amino acids, four ormore amino acids, five or more amino acids, and ten or more amino acidsto the reference enhanced targeting domain on which the non-conservativeenhanced targeting domain variant is based. A non-conservative enhancedtargeting domain variant may substitute one or more amino acids, two ormore amino acids, three or more amino acids, four or more amino acids,five or more amino acids, ten or more amino acids, 20 or more aminoacids, 30 or more amino acids, 40 or more amino acids, 50 or more aminoacids, 100 or more amino acids, 200 or more amino acids, 300 or moreamino acids, 400 or more amino acids, or 500 or more amino acids fromthe reference enhanced targeting domain on which the non-conservativeenhanced targeting domain variant is based. A non-conservative enhancedtargeting domain variant can also substitute at least 10 contiguousamino acids, at least 15 contiguous amino acids, at least 20 contiguousamino acids, or at least 25 contiguous amino acids from the referenceenhanced targeting domain on which the non-conservative enhancedtargeting domain variant is based, that possess at least 50% amino acididentity, 65% amino acid identity, 75% amino acid identity, 85% aminoacid identity or 95% amino acid identity to the reference enhancedtargeting domain on which the non-conservative enhanced targeting domainvariant is based. Non-limiting examples of a non-conservative enhancedtargeting domain variant include, e.g., non-conservative glucogen-likepeptide variants, non-conservative PACAP region variants,non-conservative CCRH region variants, non-conservative PTH variants,non-conservative IL-6 variants, non-conservative neuroleukin variants,non-conservative IGF-1 variants, and non-conservative NT-3 regionvariants.

As used herein, the term “enhanced targeting domain chimeric” means apolypeptide comprising at least a portion of an enhanced targetingdomain and at least a portion of at least one other polypeptide to forman enhanced targeting domain with at least one property different fromthe reference enhanced targeting domain, with the proviso that thisenhanced targeting domain chimeric is still capable of selectivelybinding to an non-Clostridial toxin receptor system present on aClostridial toxin target cell and initiate the overall internalizationmechanism whereby a Clostridial toxin intoxicates a target cell.

As used herein, the term “active enhanced targeting domain fragment”means any of a variety of enhanced targeting domain fragments can beuseful in aspects of the present invention with the proviso that theseactive fragments are still capable of selectively binding to annon-Clostridial toxin receptor system present on a Clostridial toxintarget cell and initiate the overall internalization mechanism whereby aClostridial toxin intoxicates a target cell. Thus, aspects of thisembodiment can include enhanced targeting domains comprising a lengthof, e.g., at least 50 amino acids, at least 100 amino acids, at least150 amino acids, at least 200 amino acids, at least 250 amino acids, atleast 300 amino acids, at least 350 amino acids, at least 400 aminoacids and at least 450 amino acids. Other aspects of this embodiment caninclude enhanced targeting domains comprising translocation domainhaving a length of, e.g., at most 50 amino acids, at most 100 aminoacids, at most 150 amino acids, at most 200 amino acids, at most 250amino acids, at most 300 amino acids, at most 350 amino acids, at most400 amino acids and at most 450 amino acids.

Any of a variety of sequence alignment methods can be used to determinepercent identity of naturally-occurring enhanced targeting domainvariants and non-naturally-occurring enhanced targeting domain variants,including, without limitation, global methods, local methods and hybridmethods, such as, e.g., segment approach methods. Protocols to determinepercent identity are routine procedures within the scope of one skilledin the art and from the teaching herein.

Thus, in an embodiment, a modified Clostridial toxin disclosed in thepresent specification comprises an enhanced targeting domain. In anaspect of this embodiment, an enhanced targeting domain comprises anaturally occurring enhanced targeting domain variant, such as, e.g., anenhanced targeting domain isoform or an enhanced targeting domainsubtype. In another aspect of this embodiment, a Clostridial toxintranslocation domain comprises a non-naturally occurring enhancedtargeting domain variant, such as, e.g., a conservative enhancedtargeting domain variant, a non-conservative enhanced targeting domainvariant, a enhanced targeting domain chimeric, an active enhancedtargeting domain fragment, or any combination thereof.

An example of an enhanced targeting domain disclosed in the presentspecification is, e.g., Glucagon like hormones, such as, e.g., secretin,glucagon-like peptides, like GLP-1 and GLP-2, pituitary adenylatecyclase activating peptide (PACAP), glicentin, glicentin-relatedpolypeptide (GRPP), oxyntomodulin (OXY), growth hormone-releasinghormone (GHRH), vasoactive intestinal peptides (VIPs), gastricinhibitory polypeptide (GIP), and calcitonin-related peptidesvisceralgut peptides, such as, e.g., gastrin, gastrin-releasing peptide (GRP,bombesin) or cholecystokinin (CCK).

Thus, in an embodiment, an enhanced binding domain derived from aglycogen-like peptide. In another embodiment, an enhanced binding domainderived from a glycogen-like peptide of SEQ ID NO: 9. In aspects of thisembodiment, an enhanced binding domain derived from a glycogen-likepeptide comprises a GRPP, a GLP-1, a GLP-2, a glucagon or anoxyntomodulin. In aspects of this embodiment, an enhanced binding domainderived from a glycogen-like peptide comprises amino acids 21-50, aminoacids 53-81, amino acids 53-89, amino acids 98-124, or amino acids146-178 of SEQ ID NO: 9.

In other aspects of this embodiment, a glycogen-like peptide comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124,or amino acids 146-178 of SEQ ID NO: 9, at least 75% amino acid identitywith amino acids 21-50, amino acids 53-81, amino acids 53-89, aminoacids 98-124, or amino acids 146-178 of SEQ ID NO: 9, at least 80% aminoacid identity with amino acids 21-50, amino acids 53-81, amino acids53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 9, atleast 85% amino acid identity with amino acids 21-50, amino acids 53-81,amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ IDNO: 9, at least 90% amino acid identity with amino acids 21-50, aminoacids 53-81, amino acids 53-89, amino acids 98-124, or amino acids146-178 of SEQ ID NO: 9 or at least 95% amino acid identity with aminoacids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124,or amino acids 146-178 of SEQ ID NO: 9. In yet other aspects of thisembodiment, a glycogen-like peptide comprises a polypeptide having,e.g., at most 70% amino acid identity with amino acids 21-50, aminoacids 53-81, amino acids 53-89, amino acids 98-124, or amino acids146-178 of SEQ ID NO: 9, at most 75% amino acid identity with aminoacids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124,or amino acids 146-178 of SEQ ID NO: 9, at most 80% amino acid identitywith amino acids 21-50, amino acids 53-81, amino acids 53-89, aminoacids 98-124, or amino acids 146-178 of SEQ ID NO: 9, at most 85% aminoacid identity with amino acids 21-50, amino acids 53-81, amino acids53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 9, atmost 90% amino acid identity with amino acids 21-50, amino acids 53-81,amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ IDNO: 9 or at most 95% amino acid identity with amino acids 21-50, aminoacids 53-81, amino acids 53-89, amino acids 98-124, or amino acids146-178 of SEQ ID NO: 9.

In other aspects of this embodiment, a glycogen-like peptide comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid substitutionsrelative to amino acids 21-50, amino acids 53-81, amino acids 53-89,amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 9. In otheraspects of this embodiment, a glycogen-like peptide comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid substitutionsrelative to amino acids 21-50, amino acids 53-81, amino acids 53-89,amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 9. In yet otheraspects of this embodiment, a glycogen-like peptide comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 21-50, amino acids 53-81, amino acids 53-89,amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 9. In otheraspects of this embodiment, a glycogen-like peptide comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 21-50, amino acids 53-81, amino acids 53-89,amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 9. In stillother aspects of this embodiment, a glycogen-like peptide comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 21-50, amino acids 53-81, amino acids 53-89,amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 9. In otheraspects of this embodiment, a glycogen-like peptide comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 21-50, amino acids 53-81, amino acids 53-89,amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 9.

In other aspects of this embodiment, a glycogen-like peptide comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to amino acids 21-50, amino acids 53-81, amino acids 53-89,amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 9. In otheraspects of this embodiment, a glycogen-like peptide comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to amino acids 21-50, amino acids 53-81, amino acids 53-89,amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 9. In yet otheraspects of this embodiment, a glycogen-like peptide comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 21-50, amino acids 53-81, amino acids 53-89, amino acids98-124, or amino acids 146-178 of SEQ ID NO: 9. In other aspects of thisembodiment, a glycogen-like peptide comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 21-50,amino acids 53-81, amino acids 53-89, amino acids 98-124, or amino acids146-178 of SEQ ID NO: 9. In still other aspects of this embodiment, aglycogen-like peptide comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 21-50, amino acids 53-81,amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ IDNO: 9. In other aspects of this embodiment, a glycogen-like peptidecomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 21-50, amino acids 53-81, amino acids 53-89,amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 9.

In another embodiment, an enhanced binding domain derived from a PACAP.In another embodiment, an enhanced binding domain derived from a PACAPof SEQ ID NO: 10. In an aspect of this embodiment, an enhanced bindingdomain derived from a PACAP comprises amino acids 132-158 of SEQ ID NO:10.

In other aspects of this embodiment, a PACAP comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 132-158of SEQ ID NO: 10, at least 75% amino acid identity with amino acids132-158 of SEQ ID NO: 10, at least 80% amino acid identity with aminoacids 132-158 of SEQ ID NO: 10, at least 85% amino acid identity withamino acids 132-158 of SEQ ID NO: 10, at least 90% amino acid identitywith amino acids 132-158 of SEQ ID NO: 10 or at least 95% amino acididentity with amino acids 132-158 of SEQ ID NO: 10. In yet other aspectsof this embodiment, a PACAP comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 132-158 of SEQ ID NO: 10,at most 75% amino acid identity with amino acids 132-158 of SEQ ID NO:10, at most 80% amino acid identity with amino acids 132-158 of SEQ IDNO: 10, at most 85% amino acid identity with amino acids 132-158 of SEQID NO: 10, at most 90% amino acid identity with amino acids 132-158 ofSEQ ID NO: 10 or at most 95% amino acid identity with amino acids132-158 of SEQ ID NO: 10.

In other aspects of this embodiment, a PACAP comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 132-158 of SEQ ID NO: 10. In other aspects of this embodiment, aPACAP comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 132-158 of SEQ ID NO: 10. In yetother aspects of this embodiment, a PACAP comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 132-158 of SEQ ID NO: 10. In other aspects of this embodiment, aPACAP comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 132-158 of SEQ ID NO: 10. In stillother aspects of this embodiment, a PACAP comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 132-158 of SEQ ID NO: 10. In other aspects of this embodiment, aPACAP comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 132-158 of SEQ ID NO: 10.

In other aspects of this embodiment, a PACAP comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 132-158 of SEQ ID NO: 10. In other aspects of this embodiment, aPACAP comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 132-158 of SEQ ID NO: 10. In yetother aspects of this embodiment, a PACAP comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids132-158 of SEQ ID NO: 10. In other aspects of this embodiment, a PACAPcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 132-158 of SEQ ID NO: 10. In still other aspectsof this embodiment, a PACAP comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 132-158 of SEQID NO: 10. In other aspects of this embodiment, a PACAP comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 132-158 of SEQ ID NO: 10.

In another embodiment, an enhanced binding domain derived from a GHRH.In another embodiment, an enhanced binding domain derived from a GHRH ofSEQ ID NO: 11. In aspects of this embodiment, an enhanced binding domainderived from a GHRH comprises amino acids 32-58 or amino acids 32-75 ofSEQ ID NO: 11.

In other aspects of this embodiment, a GHRH comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 32-58 oramino acids 32-75 of SEQ ID NO: 11, at least 75% amino acid identitywith amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 11, at least80% amino acid identity with amino acids 32-58 or amino acids 32-75 ofSEQ ID NO: 11, at least 85% amino acid identity with amino acids 32-58or amino acids 32-75 of SEQ ID NO: 11, at least 90% amino acid identitywith amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 11 or at least95% amino acid identity with amino acids 32-58 or amino acids 32-75 ofSEQ ID NO: 11. In yet other aspects of this embodiment, a GHRH comprisesa polypeptide having, e.g., at most 70% amino acid identity with aminoacids 32-58 or amino acids 32-75 of SEQ ID NO: 11, at most 75% aminoacid identity with amino acids 32-58 or amino acids 32-75 of SEQ ID NO:11, at most 80% amino acid identity with amino acids 32-58 or aminoacids 32-75 of SEQ ID NO: 11, at most 85% amino acid identity with aminoacids 32-58 or amino acids 32-75 of SEQ ID NO: 11, at most 90% aminoacid identity with amino acids 32-58 or amino acids 32-75 of SEQ ID NO:11 or at most 95% amino acid identity with amino acids 32-58 or aminoacids 32-75 of SEQ ID NO: 11.

In other aspects of this embodiment, a GHRH comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 32-58 or amino acids 32-75 of SEQ ID NO: 11. In other aspects ofthis embodiment, a GHRH comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 32-58 oramino acids 32-75 of SEQ ID NO: 11. In yet other aspects of thisembodiment, a GHRH comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 32-58 or amino acids 32-75of SEQ ID NO: 11. In other aspects of this embodiment, a GHRH comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 11. Instill other aspects of this embodiment, a GHRH comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 32-58 or amino acids 32-75 of SEQ ID NO: 11. In other aspects ofthis embodiment, a GHRH comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 32-58 oramino acids 32-75 of SEQ ID NO: 11.

In other aspects of this embodiment, a GHRH comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 32-58 or amino acids 32-75 of SEQ ID NO: 11. In other aspects ofthis embodiment, a GHRH comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 32-58 oramino acids 32-75 of SEQ ID NO: 11. In yet other aspects of thisembodiment, a GHRH comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 32-58 or amino acids 32-75of SEQ ID NO: 11. In other aspects of this embodiment, a GHRH comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 32-58 or amino acids 32-75 of SEQ ID NO: 11. In still otheraspects of this embodiment, a GHRH comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 32-58 or aminoacids 32-75 of SEQ ID NO: 11. In other aspects of this embodiment, aGHRH comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 32-58 or amino acids 32-75 of SEQ IDNO: 11.

In another embodiment, an enhanced binding domain derived from a VIP1.In another embodiment, an enhanced binding domain derived from a VIP1 ofSEQ ID NO: 12. In aspects of this embodiment, an enhanced binding domainderived from a VIP1 comprises amino acids 81-107 or amino acids 125-151of SEQ ID NO: 12.

In other aspects of this embodiment, a VIP1 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 81-107or amino acids 125-151 of SEQ ID NO: 12, at least 75% amino acididentity with amino acids 81-107 or amino acids 125-151 of SEQ ID NO:12, at least 80% amino acid identity with amino acids 81-107 or aminoacids 125-151 of SEQ ID NO: 12, at least 85% amino acid identity withamino acids 81-107 or amino acids 125-151 of SEQ ID NO: 12, at least 90%amino acid identity with amino acids 81-107 or amino acids 125-151 ofSEQ ID NO: 12 or at least 95% amino acid identity with amino acids81-107 or amino acids 125-151 of SEQ ID NO: 12. In yet other aspects ofthis embodiment, a VIP1 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 81-107 or amino acids 125-151of SEQ ID NO: 12, at most 75% amino acid identity with amino acids81-107 or amino acids 125-151 of SEQ ID NO: 12, at most 80% amino acididentity with amino acids 81-107 or amino acids 125-151 of SEQ ID NO:12, at most 85% amino acid identity with amino acids 81-107 or aminoacids 125-151 of SEQ ID NO: 12, at most 90% amino acid identity withamino acids 81-107 or amino acids 125-151 of SEQ ID NO: 12 or at most95% amino acid identity with amino acids 81-107 or amino acids 125-151of SEQ ID NO: 12.

In other aspects of this embodiment, a VIP1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 81-107 or amino acids 125-151 of SEQ ID NO: 12. In other aspectsof this embodiment, a VIP1 comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 81-107or amino acids 125-151 of SEQ ID NO: 12. In yet other aspects of thisembodiment, a VIP1 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 81-107 or amino acids125-151 of SEQ ID NO: 12. In other aspects of this embodiment, a VIP1comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 81-107 or amino acids 125-151 of SEQID NO: 12. In still other aspects of this embodiment, a VIP1 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 12.In other aspects of this embodiment, a VIP1 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 81-107 or amino acids 125-151 of SEQ ID NO: 12.

In other aspects of this embodiment, a VIP1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 81-107 or amino acids 125-151 of SEQ ID NO: 12. In other aspectsof this embodiment, a VIP1 comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 81-107 oramino acids 125-151 of SEQ ID NO: 12. In yet other aspects of thisembodiment, a VIP1 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 81-107 or amino acids125-151 of SEQ ID NO: 12. In other aspects of this embodiment, a VIP1comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 12.In still other aspects of this embodiment, a VIP1 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 81-107 or amino acids 125-151 of SEQ ID NO: 12. In otheraspects of this embodiment, a VIP1 comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 81-107 or aminoacids 125-151 of SEQ ID NO: 12.

In another embodiment, an enhanced binding domain derived from a VIP2.In another embodiment, an enhanced binding domain derived from a VIP2 ofSEQ ID NO: 13. In aspects of this embodiment, an enhanced binding domainderived from a VIP2 comprises amino acids 81-107 or amino acids 124-150of SEQ ID NO: 13.

In other aspects of this embodiment, a VIP2 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 81-107or amino acids 124-150 of SEQ ID NO: 13, at least 75% amino acididentity with amino acids 81-107 or amino acids 124-150 of SEQ ID NO:13, at least 80% amino acid identity with amino acids 81-107 or aminoacids 124-150 of SEQ ID NO: 13, at least 85% amino acid identity withamino acids 81-107 or amino acids 124-150 of SEQ ID NO: 13, at least 90%amino acid identity with amino acids 81-107 or amino acids 124-150 ofSEQ ID NO: 13 or at least 95% amino acid identity with amino acids81-107 or amino acids 124-150 of SEQ ID NO: 13. In yet other aspects ofthis embodiment, a VIP2 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 81-107 or amino acids 124-150of SEQ ID NO: 13, at most 75% amino acid identity with amino acids81-107 or amino acids 124-150 of SEQ ID NO: 13, at most 80% amino acididentity with amino acids 81-107 or amino acids 124-150 of SEQ ID NO:13, at most 85% amino acid identity with amino acids 81-107 or aminoacids 124-150 of SEQ ID NO: 13, at most 90% amino acid identity withamino acids 81-107 or amino acids 124-150 of SEQ ID NO: 13 or at most95% amino acid identity with amino acids 81-107 or amino acids 124-150of SEQ ID NO: 13.

In other aspects of this embodiment, a VIP2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 81-107 or amino acids 124-150 of SEQ ID NO: 13. In other aspectsof this embodiment, a VIP2 comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 81-107or amino acids 124-150 of SEQ ID NO: 13. In yet other aspects of thisembodiment, a VIP2 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 81-107 or amino acids124-150 of SEQ ID NO: 13. In other aspects of this embodiment, a VIP2comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 81-107 or amino acids 124-150 of SEQID NO: 13. In still other aspects of this embodiment, a VIP2 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 13.In other aspects of this embodiment, a VIP2 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 81-107 or amino acids 124-150 of SEQ ID NO: 13.

In other aspects of this embodiment, a VIP2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 81-107 or amino acids 124-150 of SEQ ID NO: 13. In other aspectsof this embodiment, a VIP2 comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 81-107 oramino acids 124-150 of SEQ ID NO: 13. In yet other aspects of thisembodiment, a VIP2 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 81-107 or amino acids124-150 of SEQ ID NO: 13. In other aspects of this embodiment, a VIP2comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 13.In still other aspects of this embodiment, a VIP2 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 81-107 or amino acids 124-150 of SEQ ID NO: 13. In otheraspects of this embodiment, a VIP2 comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 81-107 or aminoacids 124-150 of SEQ ID NO: 13.

In another embodiment, an enhanced binding domain derived from a GIP. Inanother embodiment, an enhanced binding domain derived from a GIP of SEQID NO: 14. In aspects of this embodiment, an enhanced binding domainderived from a GIP comprises amino acids 52-78 or amino acids 52-93 ofSEQ ID NO: 14.

In other aspects of this embodiment, a GIP comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 52-78 oramino acids 52-93 of SEQ ID NO: 14, at least 75% amino acid identitywith amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 14, at least80% amino acid identity with amino acids 52-78 or amino acids 52-93 ofSEQ ID NO: 14, at least 85% amino acid identity with amino acids 52-78or amino acids 52-93 of SEQ ID NO: 14, at least 90% amino acid identitywith amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 14 or at least95% amino acid identity with amino acids 52-78 or amino acids 52-93 ofSEQ ID NO: 14. In yet other aspects of this embodiment, a GIP comprisesa polypeptide having, e.g., at most 70% amino acid identity with aminoacids 52-78 or amino acids 52-93 of SEQ ID NO: 14, at most 75% aminoacid identity with amino acids 52-78 or amino acids 52-93 of SEQ ID NO:14, at most 80% amino acid identity with amino acids 52-78 or aminoacids 52-93 of SEQ ID NO: 14, at most 85% amino acid identity with aminoacids 52-78 or amino acids 52-93 of SEQ ID NO: 14, at most 90% aminoacid identity with amino acids 52-78 or amino acids 52-93 of SEQ ID NO:14 or at most 95% amino acid identity with amino acids 52-78 or aminoacids 52-93 of SEQ ID NO: 14.

In other aspects of this embodiment, a GIP comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 52-78 or amino acids 52-93 of SEQ ID NO: 14. In other aspects ofthis embodiment, a GIP comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 52-78 oramino acids 52-93 of SEQ ID NO: 14. In yet other aspects of thisembodiment, a GIP comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 52-78 or amino acids 52-93of SEQ ID NO: 14. In other aspects of this embodiment, a GIP comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 14. Instill other aspects of this embodiment, a GIP comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 52-78 or amino acids 52-93 of SEQ ID NO: 14. In other aspects ofthis embodiment, a GIP comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 52-78 oramino acids 52-93 of SEQ ID NO: 14.

In other aspects of this embodiment, a GIP comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 52-78 or amino acids 52-93 of SEQ ID NO: 14. In other aspects ofthis embodiment, a GIP comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 52-78 oramino acids 52-93 of SEQ ID NO: 14. In yet other aspects of thisembodiment, a GIP comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 52-78 or amino acids 52-93of SEQ ID NO: 14. In other aspects of this embodiment, a GIP comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 52-78 or amino acids 52-93 of SEQ ID NO: 14. In still otheraspects of this embodiment, a GIP comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 52-78 or aminoacids 52-93 of SEQ ID NO: 14. In other aspects of this embodiment, a GIPcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 14.

In another embodiment, an enhanced binding domain derived from aSecretin. In another embodiment, an enhanced binding domain derived froma Secretin of SEQ ID NO: 15. In an aspect of this embodiment, anenhanced binding domain derived from a Secretin comprises amino acids28-54 of SEQ ID NO: 15.

In other aspects of this embodiment, a Secretin comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 28-54 ofSEQ ID NO: 15, at least 75% amino acid identity with amino acids 28-54of SEQ ID NO: 15, at least 80% amino acid identity with amino acids28-54 of SEQ ID NO: 15, at least 85% amino acid identity with aminoacids 28-54 of SEQ ID NO: 15, at least 90% amino acid identity withamino acids 28-54 of SEQ ID NO: 15 or at least 95% amino acid identitywith amino acids 28-54 of SEQ ID NO: 15. In yet other aspects of thisembodiment, a Secretin comprises a polypeptide having, e.g., at most 70%amino acid identity with amino acids 28-54 of SEQ ID NO: 15, at most 75%amino acid identity with amino acids 28-54 of SEQ ID NO: 15, at most 80%amino acid identity with amino acids 28-54 of SEQ ID NO: 15, at most 85%amino acid identity with amino acids 28-54 of SEQ ID NO: 15, at most 90%amino acid identity with amino acids 28-54 of SEQ ID NO: 15 or at most95% amino acid identity with amino acids 28-54 of SEQ ID NO: 15.

In other aspects of this embodiment, a Secretin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 28-54 of SEQ ID NO: 15. In other aspects of this embodiment, aSecretin comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 28-54 of SEQ ID NO: 15. In yetother aspects of this embodiment, a Secretin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 28-54 of SEQ ID NO: 15. In other aspects of this embodiment, aSecretin comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 28-54 of SEQ ID NO: 15. In still otheraspects of this embodiment, a Secretin comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids 28-54of SEQ ID NO: 15. In other aspects of this embodiment, a Secretincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 28-54 of SEQ ID NO: 15.

In other aspects of this embodiment, a Secretin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 28-54 of SEQ ID NO: 15. In other aspects of this embodiment, aSecretin comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 28-54 of SEQ ID NO: 15. In yetother aspects of this embodiment, a Secretin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids28-54 of SEQ ID NO: 15. In other aspects of this embodiment, a Secretincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 28-54 of SEQ ID NO: 15. In still other aspectsof this embodiment, a Secretin comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 28-54 of SEQ IDNO: 15. In other aspects of this embodiment, a Secretin comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 28-54 of SEQ ID NO: 15.

In another embodiment, an enhanced binding domain derived from aGastrin. In another embodiment, an enhanced binding domain derived froma Gastrin of SEQ ID NO: 16. In aspects of this embodiment, an enhancedbinding domain derived from a Gastrin comprises amino acids 76-92 oramino acids 59-92 of SEQ ID NO: 16.

In other aspects of this embodiment, a Gastrin comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 76-92 oramino acids 59-92 of SEQ ID NO: 16, at least 75% amino acid identitywith amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 16, at least80% amino acid identity with amino acids 76-92 or amino acids 59-92 ofSEQ ID NO: 16, at least 85% amino acid identity with amino acids 76-92or amino acids 59-92 of SEQ ID NO: 16, at least 90% amino acid identitywith amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 16 or at least95% amino acid identity with amino acids 76-92 or amino acids 59-92 ofSEQ ID NO: 16. In yet other aspects of this embodiment, a Gastrincomprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 16, at most75% amino acid identity with amino acids 76-92 or amino acids 59-92 ofSEQ ID NO: 16, at most 80% amino acid identity with amino acids 76-92 oramino acids 59-92 of SEQ ID NO: 16, at most 85% amino acid identity withamino acids 76-92 or amino acids 59-92 of SEQ ID NO: 16, at most 90%amino acid identity with amino acids 76-92 or amino acids 59-92 of SEQID NO: 16 or at most 95% amino acid identity with amino acids 76-92 oramino acids 59-92 of SEQ ID NO: 16.

In other aspects of this embodiment, a Gastrin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 76-92 or amino acids 59-92 of SEQ ID NO: 16. In other aspects ofthis embodiment, a Gastrin comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 76-92 oramino acids 59-92 of SEQ ID NO: 16. In yet other aspects of thisembodiment, a Gastrin comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 76-92 or amino acids 59-92of SEQ ID NO: 16. In other aspects of this embodiment, a Gastrincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 76-92 or amino acids 59-92 of SEQ IDNO: 16. In still other aspects of this embodiment, a Gastrin comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 16. Inother aspects of this embodiment, a Gastrin comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 76-92 or amino acids 59-92 of SEQ ID NO: 16.

In other aspects of this embodiment, a Gastrin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 76-92 or amino acids 59-92 of SEQ ID NO: 16. In other aspects ofthis embodiment, a Gastrin comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 76-92 oramino acids 59-92 of SEQ ID NO: 16. In yet other aspects of thisembodiment, a Gastrin comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 76-92 or amino acids 59-92of SEQ ID NO: 16. In other aspects of this embodiment, a Gastrincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 16. Instill other aspects of this embodiment, a Gastrin comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 76-92 or amino acids 59-92 of SEQ ID NO: 16. In otheraspects of this embodiment, a Gastrin comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to amino acids 76-92 oramino acids 59-92 of SEQ ID NO: 16.

In another embodiment, an enhanced binding domain derived from a GRP. Inanother embodiment, an enhanced binding domain derived from a GRP of SEQID NO: 17. In aspects of this embodiment, an enhanced binding domainderived from a GRP comprises amino acids 41-50 or amino acids 24-50 ofSEQ ID NO: 17.

In other aspects of this embodiment, a GRP comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 41-50 oramino acids 24-50 of SEQ ID NO: 17, at least 75% amino acid identitywith amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 17, at least80% amino acid identity with amino acids 41-50 or amino acids 24-50 ofSEQ ID NO: 17, at least 85% amino acid identity with amino acids 41-50or amino acids 24-50 of SEQ ID NO: 17, at least 90% amino acid identitywith amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 17 or at least95% amino acid identity with amino acids 41-50 or amino acids 24-50 ofSEQ ID NO: 17. In yet other aspects of this embodiment, a GRP comprisesa polypeptide having, e.g., at most 70% amino acid identity with aminoacids 41-50 or amino acids 24-50 of SEQ ID NO: 17, at most 75% aminoacid identity with amino acids 41-50 or amino acids 24-50 of SEQ ID NO:17, at most 80% amino acid identity with amino acids 41-50 or aminoacids 24-50 of SEQ ID NO: 17, at most 85% amino acid identity with aminoacids 41-50 or amino acids 24-50 of SEQ ID NO: 17, at most 90% aminoacid identity with amino acids 41-50 or amino acids 24-50 of SEQ ID NO:17 or at most 95% amino acid identity with amino acids 41-50 or aminoacids 24-50 of SEQ ID NO: 17.

In other aspects of this embodiment, a GRP comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 41-50 or amino acids 24-50 of SEQ ID NO: 17. In other aspects ofthis embodiment, a GRP comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 41-50 oramino acids 24-50 of SEQ ID NO: 17. In yet other aspects of thisembodiment, a GRP comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 41-50 or amino acids 24-50of SEQ ID NO: 17. In other aspects of this embodiment, a GRP comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 17. Instill other aspects of this embodiment, a GRP comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 41-50 or amino acids 24-50 of SEQ ID NO: 17. In other aspects ofthis embodiment, a GRP comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 41-50 oramino acids 24-50 of SEQ ID NO: 17.

In other aspects of this embodiment, a GRP comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 41-50 or amino acids 24-50 of SEQ ID NO: 17. In other aspects ofthis embodiment, a GRP comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 41-50 oramino acids 24-50 of SEQ ID NO: 17. In yet other aspects of thisembodiment, a GRP comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 41-50 or amino acids 24-50of SEQ ID NO: 17. In other aspects of this embodiment, a GRP comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 41-50 or amino acids 24-50 of SEQ ID NO: 17. In still otheraspects of this embodiment, a GRP comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 41-50 or aminoacids 24-50 of SEQ ID NO: 17. In other aspects of this embodiment, a GRPcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 17.

In another embodiment, an enhanced binding domain derived from a CCK. Inanother embodiment, an enhanced binding domain derived from a CCK of SEQID NO: 18. In an aspect of this embodiment, an enhanced binding domainderived from a CCK comprises amino acids 99-112 of SEQ ID NO: 18.

In other aspects of this embodiment, a CCK comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 99-112of SEQ ID NO: 18, at least 75% amino acid identity with amino acids99-112 of SEQ ID NO: 18, at least 80% amino acid identity with aminoacids 99-112 of SEQ ID NO: 18, at least 85% amino acid identity withamino acids 99-112 of SEQ ID NO: 18, at least 90% amino acid identitywith amino acids 99-112 of SEQ ID NO: 18 or at least 95% amino acididentity with amino acids 99-112 of SEQ ID NO: 18. In yet other aspectsof this embodiment, a CCK comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 99-112 of SEQ ID NO: 18, atmost 75% amino acid identity with amino acids 99-112 of SEQ ID NO: 18,at most 80% amino acid identity with amino acids 99-112 of SEQ ID NO:18, at most 85% amino acid identity with amino acids 99-112 of SEQ IDNO: 18, at most 90% amino acid identity with amino acids 99-112 of SEQID NO: 18 or at most 95% amino acid identity with amino acids 99-112 ofSEQ ID NO: 18.

In other aspects of this embodiment, a CCK comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 99-112 of SEQ ID NO: 18. In other aspects of this embodiment, aCCK comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 99-112 of SEQ ID NO: 18. In yetother aspects of this embodiment, a CCK comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids 99-112of SEQ ID NO: 18. In other aspects of this embodiment, a CCK comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 99-112 of SEQ ID NO: 18. In still other aspectsof this embodiment, a CCK comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 99-112 ofSEQ ID NO: 18. In other aspects of this embodiment, a CCK comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 99-112 of SEQ ID NO: 18.

In other aspects of this embodiment, a CCK comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 99-112 of SEQ ID NO: 18. In other aspects of this embodiment, aCCK comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 99-112 of SEQ ID NO: 18. In yetother aspects of this embodiment, a CCK comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 99-112 ofSEQ ID NO: 18. In other aspects of this embodiment, a CCK comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 99-112 of SEQ ID NO: 18. In still other aspects of thisembodiment, a CCK comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 99-112 of SEQ ID NO: 18. Inother aspects of this embodiment, a CCK comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to amino acids 99-112 ofSEQ ID NO: 18.

Another example of an enhanced targeting domain disclosed in the presentspecification is, e.g., neurohormones, such as, e.g.,corticotropin-releasing hormone (CCRH) or parathyroid hormone (PTH).

Thus, in an embodiment, an enhanced binding domain derived from a CCRH.In another embodiment, an enhanced binding domain derived from a CCRH ofSEQ ID NO: 19. In aspects of this embodiment, an enhanced binding domainderived from a CCRH comprises amino acids 159-193 or amino acids 154-194of SEQ ID NO: 19.

In other aspects of this embodiment, a CCRH comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 159-193or amino acids 154-194 of SEQ ID NO: 19, at least 75% amino acididentity with amino acids 159-193 or amino acids 154-194 of SEQ ID NO:19, at least 80% amino acid identity with amino acids 159-193 or aminoacids 154-194 of SEQ ID NO: 19, at least 85% amino acid identity withamino acids 159-193 or amino acids 154-194 of SEQ ID NO: 19, at least90% amino acid identity with amino acids 159-193 or amino acids 154-194of SEQ ID NO: 19 or at least 95% amino acid identity with amino acids159-193 or amino acids 154-194 of SEQ ID NO: 19. In yet other aspects ofthis embodiment, a CCRH comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 159-193 or amino acids 154-194of SEQ ID NO: 19, at most 75% amino acid identity with amino acids159-193 or amino acids 154-194 of SEQ ID NO: 19, at most 80% amino acididentity with amino acids 159-193 or amino acids 154-194 of SEQ ID NO:19, at most 85% amino acid identity with amino acids 159-193 or aminoacids 154-194 of SEQ ID NO: 19, at most 90% amino acid identity withamino acids 159-193 or amino acids 154-194 of SEQ ID NO: 19 or at most95% amino acid identity with amino acids 159-193 or amino acids 154-194of SEQ ID NO: 19.

In other aspects of this embodiment, a CCRH comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 159-193 or amino acids 154-194 of SEQ ID NO: 19. In other aspectsof this embodiment, a CCRH comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 159-193or amino acids 154-194 of SEQ ID NO: 19. In yet other aspects of thisembodiment, a CCRH comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 159-193 or amino acids154-194 of SEQ ID NO: 19. In other aspects of this embodiment, a CCRHcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 159-193 or amino acids 154-194 of SEQID NO: 19. In still other aspects of this embodiment, a CCRH comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 19.In other aspects of this embodiment, a CCRH comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 159-193 or amino acids 154-194 of SEQ ID NO: 19.

In other aspects of this embodiment, a CCRH comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 159-193 or amino acids 154-194 of SEQ ID NO: 19. In other aspectsof this embodiment, a CCRH comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 159-193 oramino acids 154-194 of SEQ ID NO: 19. In yet other aspects of thisembodiment, a CCRH comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 159-193 or amino acids154-194 of SEQ ID NO: 19. In other aspects of this embodiment, a CCRHcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 19.In still other aspects of this embodiment, a CCRH comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 159-193 or amino acids 154-194 of SEQ ID NO: 19. In otheraspects of this embodiment, a CCRH comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 159-193 or aminoacids 154-194 of SEQ ID NO: 19.

In another embodiment, an enhanced binding domain derived from a PTH. Inanother embodiment, an enhanced binding domain derived from a PTH of SEQID NO: 20. In aspects of this embodiment, an enhanced binding domainderived from a PTH comprises amino acids 35-70 or amino acids 145-177 ofSEQ ID NO: 20.

In other aspects of this embodiment, a PTH comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 35-70 oramino acids 145-177 of SEQ ID NO: 20, at least 75% amino acid identitywith amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 20, at least80% amino acid identity with amino acids 35-70 or amino acids 145-177 ofSEQ ID NO: 20, at least 85% amino acid identity with amino acids 35-70or amino acids 145-177 of SEQ ID NO: 20, at least 90% amino acididentity with amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 20or at least 95% amino acid identity with amino acids 35-70 or aminoacids 145-177 of SEQ ID NO: 20. In yet other aspects of this embodiment,a PTH comprises a polypeptide having, e.g., at most 70% amino acididentity with amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 20,at most 75% amino acid identity with amino acids 35-70 or amino acids145-177 of SEQ ID NO: 20, at most 80% amino acid identity with aminoacids 35-70 or amino acids 145-177 of SEQ ID NO: 20, at most 85% aminoacid identity with amino acids 35-70 or amino acids 145-177 of SEQ IDNO: 20, at most 90% amino acid identity with amino acids 35-70 or aminoacids 145-177 of SEQ ID NO: 20 or at most 95% amino acid identity withamino acids 35-70 or amino acids 145-177 of SEQ ID NO: 20.

In other aspects of this embodiment, a PTH comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 35-70 or amino acids 145-177 of SEQ ID NO: 20. In other aspects ofthis embodiment, a PTH comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 35-70 oramino acids 145-177 of SEQ ID NO: 20. In yet other aspects of thisembodiment, a PTH comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 35-70 or amino acids145-177 of SEQ ID NO: 20. In other aspects of this embodiment, a PTHcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 35-70 or amino acids 145-177 of SEQ IDNO: 20. In still other aspects of this embodiment, a PTH comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 20.In other aspects of this embodiment, a PTH comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 35-70 or amino acids 145-177 of SEQ ID NO: 20.

In other aspects of this embodiment, a PTH comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 35-70 or amino acids 145-177 of SEQ ID NO: 20. In other aspects ofthis embodiment, a PTH comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 35-70 oramino acids 145-177 of SEQ ID NO: 20. In yet other aspects of thisembodiment, a PTH comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 35-70 or amino acids145-177 of SEQ ID NO: 20. In other aspects of this embodiment, a PTHcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 20.In still other aspects of this embodiment, a PTH comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids35-70 or amino acids 145-177 of SEQ ID NO: 20. In other aspects of thisembodiment, a PTH comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 35-70 or amino acids145-177 of SEQ ID NO: 20.

Another example of an enhanced targeting domain disclosed in the presentspecification is, e.g., neuroregulatory cytokines, such as, e.g.,ciliary neurotrophic factor (CNTF), glycophorin-A (GPA), leukemiainhibitory factor (LIF), also known as cholinergic differentiationfactor (CDF), interleukins (ILs), like IL1, IL2, IL6, IL8 and IL10,onostatin M, cardiotrophin-1 (CT-1), cardiotrophin-like cytokine (CLC),or neuroleukin, also known as glucose phosphate isomerase (GPI),autocrine motility factor (AMF), maturation and differentiation factor(MF).

Thus, in an embodiment, an enhanced binding domain derived from a CNTF.In another embodiment, an enhanced binding domain derived from a CNTF ofSEQ ID NO: 21. In aspects of this embodiment, a CNTF comprises apolypeptide having, e.g., at least 70% amino acid identity with theamino acid sequence of SEQ ID NO: 21, at least 75% amino acid identitywith the amino acid sequence of SEQ ID NO: 21, at least 80% amino acididentity with the amino acid sequence of SEQ ID NO: 21, at least 85%amino acid identity with the amino acid sequence of SEQ ID NO: 21, atleast 90% amino acid identity with the amino acid sequence of SEQ ID NO:21 or at least 95% amino acid identity with the amino acid sequence ofSEQ ID NO: 21. In yet other aspects of this embodiment, a CNTF comprisesa polypeptide having, e.g., at most 70% amino acid identity with theamino acid sequence of SEQ ID NO: 21, at most 75% amino acid identitywith the amino acid sequence of SEQ ID NO: 21, at most 80% amino acididentity with the amino acid sequence of SEQ ID NO: 21, at most 85%amino acid identity with the amino acid sequence of SEQ ID NO: 21, atmost 90% amino acid identity with the amino acid sequence of SEQ ID NO:21 or at most 95% amino acid identity with the amino acid sequence ofSEQ ID NO: 21.

In other aspects of this embodiment, a CNTF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to theamino acid sequence of SEQ ID NO: 21. In other aspects of thisembodiment, a CNTF comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to the amino acid sequence of SEQ IDNO: 21. In yet other aspects of this embodiment, a CNTF comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to the amino acid sequence of SEQ ID NO: 21. In other aspectsof this embodiment, a CNTF comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to the amino acid sequenceof SEQ ID NO: 21. In still other aspects of this embodiment, a CNTFcomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to the amino acid sequence of SEQ ID NO: 21. In otheraspects of this embodiment, a CNTF comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to the amino acid sequenceof SEQ ID NO: 21.

In other aspects of this embodiment, a CNTF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to the aminoacid sequence of SEQ ID NO: 21. In other aspects of this embodiment, aCNTF comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to the amino acid sequence of SEQ ID NO: 21. Inyet other aspects of this embodiment, a CNTF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to the aminoacid sequence of SEQ ID NO: 21. In other aspects of this embodiment, aCNTF comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to the amino acid sequence of SEQ ID NO: 21. In stillother aspects of this embodiment, a CNTF comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to the amino acidsequence of SEQ ID NO: 21. In other aspects of this embodiment, a CNTFcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to the amino acid sequence of SEQ ID NO: 21.

In another embodiment, an enhanced binding domain derived from a GPA. Inanother embodiment, an enhanced binding domain derived from a GPA of SEQID NO: 22. In aspects of this embodiment, a GPA comprises a polypeptidehaving, e.g., at least 70% amino acid identity with the amino acidsequence of SEQ ID NO: 22, at least 75% amino acid identity with theamino acid sequence of SEQ ID NO: 22, at least 80% amino acid identitywith the amino acid sequence of SEQ ID NO: 22, at least 85% amino acididentity with the amino acid sequence of SEQ ID NO: 22, at least 90%amino acid identity with the amino acid sequence of SEQ ID NO: 22 or atleast 95% amino acid identity with the amino acid sequence of SEQ ID NO:22. In yet other aspects of this embodiment, a GPA comprises apolypeptide having, e.g., at most 70% amino acid identity with the aminoacid sequence of SEQ ID NO: 22, at most 75% amino acid identity with theamino acid sequence of SEQ ID NO: 22, at most 80% amino acid identitywith the amino acid sequence of SEQ ID NO: 22, at most 85% amino acididentity with the amino acid sequence of SEQ ID NO: 22, at most 90%amino acid identity with the amino acid sequence of SEQ ID NO: 22 or atmost 95% amino acid identity with the amino acid sequence of SEQ ID NO:22.

In other aspects of this embodiment, a GPA comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to theamino acid sequence of SEQ ID NO: 22. In other aspects of thisembodiment, a GPA comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to the amino acid sequence of SEQ IDNO: 22. In yet other aspects of this embodiment, a GPA comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to the amino acid sequence of SEQ ID NO: 22. In other aspectsof this embodiment, a GPA comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to the amino acid sequenceof SEQ ID NO: 22. In still other aspects of this embodiment, a GPAcomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to the amino acid sequence of SEQ ID NO: 22. In otheraspects of this embodiment, a GPA comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to the amino acid sequenceof SEQ ID NO: 22.

In other aspects of this embodiment, a GPA comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to the aminoacid sequence of SEQ ID NO: 22. In other aspects of this embodiment, aGPA comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to the amino acid sequence of SEQ ID NO: 22. Inyet other aspects of this embodiment, a GPA comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to the aminoacid sequence of SEQ ID NO: 22. In other aspects of this embodiment, aGPA comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to the amino acid sequence of SEQ ID NO: 22. In stillother aspects of this embodiment, a GPA comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to the amino acidsequence of SEQ ID NO: 22. In other aspects of this embodiment, a GPAcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to the amino acid sequence of SEQ ID NO: 22.

In another embodiment, an enhanced binding domain derived from a LIF. Inanother embodiment, an enhanced binding domain derived from a LIF of SEQID NO: 23. In aspects of this embodiment, a LIF comprises a polypeptidehaving, e.g., at least 70% amino acid identity with the amino acidsequence of SEQ ID NO: 23, at least 75% amino acid identity with theamino acid sequence of SEQ ID NO: 23, at least 80% amino acid identitywith the amino acid sequence of SEQ ID NO: 23, at least 85% amino acididentity with the amino acid sequence of SEQ ID NO: 23, at least 90%amino acid identity with the amino acid sequence of SEQ ID NO: 23 or atleast 95% amino acid identity with the amino acid sequence of SEQ ID NO:23. In yet other aspects of this embodiment, a LIF comprises apolypeptide having, e.g., at most 70% amino acid identity with the aminoacid sequence of SEQ ID NO: 23, at most 75% amino acid identity with theamino acid sequence of SEQ ID NO: 23, at most 80% amino acid identitywith the amino acid sequence of SEQ ID NO: 23, at most 85% amino acididentity with the amino acid sequence of SEQ ID NO: 23, at most 90%amino acid identity with the amino acid sequence of SEQ ID NO: 23 or atmost 95% amino acid identity with the amino acid sequence of SEQ ID NO:23.

In other aspects of this embodiment, a LIF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to theamino acid sequence of SEQ ID NO: 23. In other aspects of thisembodiment, a LIF comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to the amino acid sequence of SEQ IDNO: 23. In yet other aspects of this embodiment, a LIF comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to the amino acid sequence of SEQ ID NO: 23. In other aspectsof this embodiment, a LIF comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to the amino acid sequenceof SEQ ID NO: 23. In still other aspects of this embodiment, a LIFcomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to the amino acid sequence of SEQ ID NO: 23. In otheraspects of this embodiment, a LIF comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to the amino acid sequenceof SEQ ID NO: 23.

In other aspects of this embodiment, a LIF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to the aminoacid sequence of SEQ ID NO: 23. In other aspects of this embodiment, aLIF comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to the amino acid sequence of SEQ ID NO: 23. Inyet other aspects of this embodiment, a LIF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to the aminoacid sequence of SEQ ID NO: 23. In other aspects of this embodiment, aLIF comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to the amino acid sequence of SEQ ID NO: 23. In stillother aspects of this embodiment, a LIF comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to the amino acidsequence of SEQ ID NO: 23. In other aspects of this embodiment, a LIFcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to the amino acid sequence of SEQ ID NO: 23.

In another embodiment, an enhanced binding domain derived from a CT-1.In another embodiment, an enhanced binding domain derived from a CT-1 ofSEQ ID NO: 24. In aspects of this embodiment, a CT-1 comprises apolypeptide having, e.g., at least 70% amino acid identity with theamino acid sequence of SEQ ID NO: 24, at least 75% amino acid identitywith the amino acid sequence of SEQ ID NO: 24, at least 80% amino acididentity with the amino acid sequence of SEQ ID NO: 24, at least 85%amino acid identity with the amino acid sequence of SEQ ID NO: 24, atleast 90% amino acid identity with the amino acid sequence of SEQ ID NO:24 or at least 95% amino acid identity with the amino acid sequence ofSEQ ID NO: 24. In yet other aspects of this embodiment, a CT-1 comprisesa polypeptide having, e.g., at most 70% amino acid identity with theamino acid sequence of SEQ ID NO: 24, at most 75% amino acid identitywith the amino acid sequence of SEQ ID NO: 24, at most 80% amino acididentity with the amino acid sequence of SEQ ID NO: 24, at most 85%amino acid identity with the amino acid sequence of SEQ ID NO: 24, atmost 90% amino acid identity with the amino acid sequence of SEQ ID NO:24 or at most 95% amino acid identity with the amino acid sequence ofSEQ ID NO: 24.

In other aspects of this embodiment, a CT-1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to theamino acid sequence of SEQ ID NO: 24. In other aspects of thisembodiment, a CT-1 comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to the amino acid sequence of SEQ IDNO: 24. In yet other aspects of this embodiment, a CT-1 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to the amino acid sequence of SEQ ID NO: 24. In other aspectsof this embodiment, a CT-1 comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to the amino acid sequenceof SEQ ID NO: 24. In still other aspects of this embodiment, a CT-1comprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to the amino acid sequence of SEQ ID NO: 24. In otheraspects of this embodiment, a CT-1 comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to the amino acid sequenceof SEQ ID NO: 24.

In other aspects of this embodiment, a CT-1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to the aminoacid sequence of SEQ ID NO: 24. In other aspects of this embodiment, aCT-1 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to the amino acid sequence of SEQ ID NO: 24. Inyet other aspects of this embodiment, a CT-1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to the aminoacid sequence of SEQ ID NO: 24. In other aspects of this embodiment, aCT-1 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to the amino acid sequence of SEQ ID NO: 24. In stillother aspects of this embodiment, a CT-1 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to the amino acidsequence of SEQ ID NO: 24. In other aspects of this embodiment, a CT-1comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to the amino acid sequence of SEQ ID NO: 24.

In another embodiment, an enhanced binding domain derived from a CLC. Inanother embodiment, an enhanced binding domain derived from a CLC of SEQID NO: 25. In aspects of this embodiment, a CLC comprises a polypeptidehaving, e.g., at least 70% amino acid identity with the amino acidsequence of SEQ ID NO: 25, at least 75% amino acid identity with theamino acid sequence of SEQ ID NO: 25, at least 80% amino acid identitywith the amino acid sequence of SEQ ID NO: 25, at least 85% amino acididentity with the amino acid sequence of SEQ ID NO: 25, at least 90%amino acid identity with the amino acid sequence of SEQ ID NO: 25 or atleast 95% amino acid identity with the amino acid sequence of SEQ ID NO:25. In yet other aspects of this embodiment, a CLC comprises apolypeptide having, e.g., at most 70% amino acid identity with the aminoacid sequence of SEQ ID NO: 25, at most 75% amino acid identity with theamino acid sequence of SEQ ID NO: 25, at most 80% amino acid identitywith the amino acid sequence of SEQ ID NO: 25, at most 85% amino acididentity with the amino acid sequence of SEQ ID NO: 25, at most 90%amino acid identity with the amino acid sequence of SEQ ID NO: 25 or atmost 95% amino acid identity with the amino acid sequence of SEQ ID NO:25.

In other aspects of this embodiment, a CLC comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to theamino acid sequence of SEQ ID NO: 25. In other aspects of thisembodiment, a CLC comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to the amino acid sequence of SEQ IDNO: 25. In yet other aspects of this embodiment, a CLC comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to the amino acid sequence of SEQ ID NO: 25. In other aspectsof this embodiment, a CLC comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to the amino acid sequenceof SEQ ID NO: 25. In still other aspects of this embodiment, a CLCcomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to the amino acid sequence of SEQ ID NO: 25. In otheraspects of this embodiment, a CLC comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to the amino acid sequenceof SEQ ID NO: 25.

In other aspects of this embodiment, a CLC comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to the aminoacid sequence of SEQ ID NO: 25. In other aspects of this embodiment, aCLC comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to the amino acid sequence of SEQ ID NO: 25. Inyet other aspects of this embodiment, a CLC comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to the aminoacid sequence of SEQ ID NO: 25. In other aspects of this embodiment, aCLC comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to the amino acid sequence of SEQ ID NO: 25. In stillother aspects of this embodiment, a CLC comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to the amino acidsequence of SEQ ID NO: 25. In other aspects of this embodiment, a CLCcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to the amino acid sequence of SEQ ID NO: 25.

In another embodiment, an enhanced binding domain derived from an IL-1.In another embodiment, an enhanced binding domain derived from an IL-1of SEQ ID NO: 26. In an aspect of this embodiment, an enhanced bindingdomain derived from an IL-1 comprises amino acids 123-265 of SEQ ID NO:26.

In other aspects of this embodiment, an IL-1 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 123-265of SEQ ID NO: 26, at least 75% amino acid identity with amino acids123-265 of SEQ ID NO: 26, at least 80% amino acid identity with aminoacids 123-265 of SEQ ID NO: 26, at least 85% amino acid identity withamino acids 123-265 of SEQ ID NO: 26, at least 90% amino acid identitywith amino acids 123-265 of SEQ ID NO: 26 or at least 95% amino acididentity with amino acids 123-265 of SEQ ID NO: 26. In yet other aspectsof this embodiment, an IL-1 comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 123-265 of SEQ ID NO: 26,at most 75% amino acid identity with amino acids 123-265 of SEQ ID NO:26, at most 80% amino acid identity with amino acids 123-265 of SEQ IDNO: 26, at most 85% amino acid identity with amino acids 123-265 of SEQID NO: 26, at most 90% amino acid identity with amino acids 123-265 ofSEQ ID NO: 26 or at most 95% amino acid identity with amino acids123-265 of SEQ ID NO: 26.

In other aspects of this embodiment, an IL-1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 123-265 of SEQ ID NO: 26. In other aspects of this embodiment, anIL-1 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 123-265 of SEQ ID NO: 26. In yetother aspects of this embodiment, an IL-1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 123-265 of SEQ ID NO: 26. In other aspects of this embodiment, anIL-1 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 123-265 of SEQ ID NO: 26. In stillother aspects of this embodiment, an IL-1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 123-265 of SEQ ID NO: 26. In other aspects of this embodiment, anIL-1 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 123-265 of SEQ ID NO: 26.

In other aspects of this embodiment, an IL-1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 123-265 of SEQ ID NO: 26. In other aspects of this embodiment, anIL-1 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 123-265 of SEQ ID NO: 26. In yetother aspects of this embodiment, an IL-1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids123-265 of SEQ ID NO: 26. In other aspects of this embodiment, an IL-1comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 123-265 of SEQ ID NO: 26. In still other aspectsof this embodiment, an IL-1 comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 123-265 of SEQID NO: 26. In other aspects of this embodiment, an IL-1 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 123-265 of SEQ ID NO: 26.

In another embodiment, an enhanced binding domain derived from an IL-2.In another embodiment, an enhanced binding domain derived from an IL-2of SEQ ID NO: 27. In an aspect of this embodiment, an enhanced bindingdomain derived from an IL-2 comprises amino acids 21-153 of SEQ ID NO:27.

In other aspects of this embodiment, an IL-2 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 21-153of SEQ ID NO: 27, at least 75% amino acid identity with amino acids21-153 of SEQ ID NO: 27, at least 80% amino acid identity with aminoacids 21-153 of SEQ ID NO: 27, at least 85% amino acid identity withamino acids 21-153 of SEQ ID NO: 27, at least 90% amino acid identitywith amino acids 21-153 of SEQ ID NO: 27 or at least 95% amino acididentity with amino acids 21-153 of SEQ ID NO: 27. In yet other aspectsof this embodiment, an IL-2 comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 21-153 of SEQ ID NO: 27,at most 75% amino acid identity with amino acids 21-153 of SEQ ID NO:27, at most 80% amino acid identity with amino acids 21-153 of SEQ IDNO: 27, at most 85% amino acid identity with amino acids 21-153 of SEQID NO: 27, at most 90% amino acid identity with amino acids 21-153 ofSEQ ID NO: 27 or at most 95% amino acid identity with amino acids 21-153of SEQ ID NO: 27.

In other aspects of this embodiment, an IL-2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 21-153 of SEQ ID NO: 27. In other aspects of this embodiment, anIL-2 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 21-153 of SEQ ID NO: 27. In yetother aspects of this embodiment, an IL-2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 21-153 of SEQ ID NO: 27. In other aspects of this embodiment, anIL-2 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 21-153 of SEQ ID NO: 27. In stillother aspects of this embodiment, an IL-2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 21-153 of SEQ ID NO: 27. In other aspects of this embodiment, anIL-2 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 21-153 of SEQ ID NO: 27.

In other aspects of this embodiment, an IL-2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 21-153 of SEQ ID NO: 27. In other aspects of this embodiment, anIL-2 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 21-153 of SEQ ID NO: 27. In yetother aspects of this embodiment, an IL-2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids21-153 of SEQ ID NO: 27. In other aspects of this embodiment, an IL-2comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 21-153 of SEQ ID NO: 27. In still other aspectsof this embodiment, an IL-2 comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 21-153 of SEQ IDNO: 27. In other aspects of this embodiment, an IL-2 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 21-153 of SEQ ID NO: 27.

In another embodiment, an enhanced binding domain derived from an IL-6.In another embodiment, an enhanced binding domain derived from an IL-6of SEQ ID NO: 28. In an aspect of this embodiment, an enhanced bindingdomain derived from an IL-6 comprises amino acids 57-210 of SEQ ID NO:28.

In other aspects of this embodiment, an IL-6 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 57-210of SEQ ID NO: 28, at least 75% amino acid identity with amino acids57-210 of SEQ ID NO: 28, at least 80% amino acid identity with aminoacids 57-210 of SEQ ID NO: 28, at least 85% amino acid identity withamino acids 57-210 of SEQ ID NO: 28, at least 90% amino acid identitywith amino acids 57-210 of SEQ ID NO: 28 or at least 95% amino acididentity with amino acids 57-210 of SEQ ID NO: 28. In yet other aspectsof this embodiment, an IL-6 comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 57-210 of SEQ ID NO: 28,at most 75% amino acid identity with amino acids 57-210 of SEQ ID NO:28, at most 80% amino acid identity with amino acids 57-210 of SEQ IDNO: 28, at most 85% amino acid identity with amino acids 57-210 of SEQID NO: 28, at most 90% amino acid identity with amino acids 57-210 ofSEQ ID NO: 28 or at most 95% amino acid identity with amino acids 57-210of SEQ ID NO: 28.

In other aspects of this embodiment, an IL-6 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 57-210 of SEQ ID NO: 28. In other aspects of this embodiment, anIL-6 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 57-210 of SEQ ID NO: 28. In yetother aspects of this embodiment, an IL-6 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 57-210 of SEQ ID NO: 28. In other aspects of this embodiment, anIL-6 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 57-210 of SEQ ID NO: 28. In stillother aspects of this embodiment, an IL-6 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 57-210 of SEQ ID NO: 28. In other aspects of this embodiment, anIL-6 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 57-210 of SEQ ID NO: 28.

In other aspects of this embodiment, an IL-6 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 57-210 of SEQ ID NO: 28. In other aspects of this embodiment, anIL-6 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 57-210 of SEQ ID NO: 28. In yetother aspects of this embodiment, an IL-6 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids57-210 of SEQ ID NO: 28. In other aspects of this embodiment, an IL-6comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 57-210 of SEQ ID NO: 28. In still other aspectsof this embodiment, an IL-6 comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 57-210 of SEQ IDNO: 28. In other aspects of this embodiment, an IL-6 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 57-210 of SEQ ID NO: 28.

In another embodiment, an enhanced binding domain derived from an IL-8.In another embodiment, an enhanced binding domain derived from an IL-8of SEQ ID NO: 29. In an aspect of this embodiment, an enhanced bindingdomain derived from an IL-8 comprises amino acids 21-99 or amino acids31-94 of SEQ ID NO: 29.

In other aspects of this embodiment, an IL-8 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 21-99 oramino acids 31-94 of SEQ ID NO: 29, at least 75% amino acid identitywith amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 29, at least80% amino acid identity with amino acids 21-99 or amino acids 31-94 ofSEQ ID NO: 29, at least 85% amino acid identity with amino acids 21-99or amino acids 31-94 of SEQ ID NO: 29, at least 90% amino acid identitywith amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 29 or at least95% amino acid identity with amino acids 21-99 or amino acids 31-94 ofSEQ ID NO: 29. In yet other aspects of this embodiment, an IL-8comprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 29, at most75% amino acid identity with amino acids 21-99 or amino acids 31-94 ofSEQ ID NO: 29, at most 80% amino acid identity with amino acids 21-99 oramino acids 31-94 of SEQ ID NO: 29, at most 85% amino acid identity withamino acids 21-99 or amino acids 31-94 of SEQ ID NO: 29, at most 90%amino acid identity with amino acids 21-99 or amino acids 31-94 of SEQID NO: 29 or at most 95% amino acid identity with amino acids 21-99 oramino acids 31-94 of SEQ ID NO: 29.

In other aspects of this embodiment, an IL-8 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 21-99 or amino acids 31-94 of SEQ ID NO: 29. In other aspects ofthis embodiment, an IL-8 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 21-99 oramino acids 31-94 of SEQ ID NO: 29. In yet other aspects of thisembodiment, an IL-8 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 21-99 or amino acids 31-94of SEQ ID NO: 29. In other aspects of this embodiment, an IL-8 comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 29. Instill other aspects of this embodiment, an IL-8 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 21-99 or amino acids 31-94 of SEQ ID NO: 29. In other aspects ofthis embodiment, an IL-8 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 21-99 oramino acids 31-94 of SEQ ID NO: 29.

In other aspects of this embodiment, an IL-8 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 21-99 or amino acids 31-94 of SEQ ID NO: 29. In other aspects ofthis embodiment, an IL-8 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 21-99 oramino acids 31-94 of SEQ ID NO: 29. In yet other aspects of thisembodiment, an IL-8 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 21-99 or amino acids 31-94of SEQ ID NO: 29. In other aspects of this embodiment, an IL-8 comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 21-99 or amino acids 31-94 of SEQ ID NO: 29. In still otheraspects of this embodiment, an IL-8 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to amino acids 21-99 oramino acids 31-94 of SEQ ID NO: 29. In other aspects of this embodiment,an IL-8 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 21-99 or amino acids 31-94 of SEQ IDNO: 29.

In another embodiment, an enhanced binding domain derived from an IL-10.In another embodiment, an enhanced binding domain derived from an IL-10of SEQ ID NO: 30. In an aspect of this embodiment, an enhanced bindingdomain derived from an IL-10 comprises amino acids 37-173 or amino acids19-178 of SEQ ID NO: 30.

In other aspects of this embodiment, an IL-10 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 37-173or amino acids 19-178 of SEQ ID NO: 30, at least 75% amino acid identitywith amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 30, at least80% amino acid identity with amino acids 37-173 or amino acids 19-178 ofSEQ ID NO: 30, at least 85% amino acid identity with amino acids 37-173or amino acids 19-178 of SEQ ID NO: 30, at least 90% amino acid identitywith amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 30 or atleast 95% amino acid identity with amino acids 37-173 or amino acids19-178 of SEQ ID NO: 30. In yet other aspects of this embodiment, anIL-10 comprises a polypeptide having, e.g., at most 70% amino acididentity with amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 30,at most 75% amino acid identity with amino acids 37-173 or amino acids19-178 of SEQ ID NO: 30, at most 80% amino acid identity with aminoacids 37-173 or amino acids 19-178 of SEQ ID NO: 30, at most 85% aminoacid identity with amino acids 37-173 or amino acids 19-178 of SEQ IDNO: 30, at most 90% amino acid identity with amino acids 37-173 or aminoacids 19-178 of SEQ ID NO: 30 or at most 95% amino acid identity withamino acids 37-173 or amino acids 19-178 of SEQ ID NO: 30.

In other aspects of this embodiment, an IL-10 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 37-173 or amino acids 19-178 of SEQ ID NO: 30. In other aspects ofthis embodiment, an IL-10 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 37-173or amino acids 19-178 of SEQ ID NO: 30. In yet other aspects of thisembodiment, an IL-10 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 37-173 or amino acids19-178 of SEQ ID NO: 30. In other aspects of this embodiment, an IL-10comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 37-173 or amino acids 19-178 of SEQ IDNO: 30. In still other aspects of this embodiment, an IL-10 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 30.In other aspects of this embodiment, an IL-10 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 37-173 or amino acids 19-178 of SEQ ID NO: 30.

In other aspects of this embodiment, an IL-10 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 37-173 or amino acids 19-178 of SEQ ID NO: 30. In other aspects ofthis embodiment, an IL-10 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 37-173 oramino acids 19-178 of SEQ ID NO: 30. In yet other aspects of thisembodiment, an IL-10 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 37-173 or amino acids19-178 of SEQ ID NO: 30. In other aspects of this embodiment, an IL-10comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 30.In still other aspects of this embodiment, an IL-10 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 37-173 or amino acids 19-178 of SEQ ID NO: 30. In otheraspects of this embodiment, an IL-10 comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to amino acids 37-173 oramino acids 19-178 of SEQ ID NO: 30.

In another embodiment, an enhanced binding domain derived from aneuroleukin. In another embodiment, an enhanced binding domain derivedfrom a neuroleukin of SEQ ID NO: 31. In aspects of this embodiment, aneuroleukin comprises a polypeptide having, e.g., at least 70% aminoacid identity with the amino acid sequence of SEQ ID NO: 31, at least75% amino acid identity with the amino acid sequence of SEQ ID NO: 31,at least 80% amino acid identity with the amino acid sequence of SEQ IDNO: 31, at least 85% amino acid identity with the amino acid sequence ofSEQ ID NO: 31, at least 90% amino acid identity with the amino acidsequence of SEQ ID NO: 31 or at least 95% amino acid identity with theamino acid sequence of SEQ ID NO: 31. In yet other aspects of thisembodiment, a neuroleukin comprises a polypeptide having, e.g., at most70% amino acid identity with the amino acid sequence of SEQ ID NO: 31,at most 75% amino acid identity with the amino acid sequence of SEQ IDNO: 31, at most 80% amino acid identity with the amino acid sequence ofSEQ ID NO: 31, at most 85% amino acid identity with the amino acidsequence of SEQ ID NO: 31, at most 90% amino acid identity with theamino acid sequence of SEQ ID NO: 31 or at most 95% amino acid identitywith the amino acid sequence of SEQ ID NO: 31.

In other aspects of this embodiment, a neuroleukin comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid substitutionsrelative to the amino acid sequence of SEQ ID NO: 31. In other aspectsof this embodiment, a neuroleukin comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to the amino acidsequence of SEQ ID NO: 31. In yet other aspects of this embodiment, aneuroleukin comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to the amino acid sequence of SEQ ID NO:31. In other aspects of this embodiment, a neuroleukin comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to the amino acid sequence of SEQ ID NO: 31. In still otheraspects of this embodiment, a neuroleukin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to the aminoacid sequence of SEQ ID NO: 31. In other aspects of this embodiment, aneuroleukin comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid additions relative to the amino acid sequence of SEQ ID NO:31.

In other aspects of this embodiment, a neuroleukin comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to the amino acid sequence of SEQ ID NO: 31. In other aspectsof this embodiment, a neuroleukin comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to the amino acid sequenceof SEQ ID NO: 31. In yet other aspects of this embodiment, a neuroleukincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to the amino acid sequence of SEQ ID NO: 31. In other aspectsof this embodiment, a neuroleukin comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to the amino acid sequence ofSEQ ID NO: 31. In still other aspects of this embodiment, a neuroleukincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to the amino acid sequence of SEQ ID NO: 31. In other aspectsof this embodiment, a neuroleukin comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to the amino acid sequence ofSEQ ID NO: 31.

In another embodiment, an enhanced binding domain derived from a VEGF.In another embodiment, an enhanced binding domain derived from a VEGF ofSEQ ID NO: 32. In aspects of this embodiment, a VEGF comprises apolypeptide having, e.g., at least 70% amino acid identity with theamino acid sequence of SEQ ID NO: 32, at least 75% amino acid identitywith the amino acid sequence of SEQ ID NO: 32, at least 80% amino acididentity with the amino acid sequence of SEQ ID NO: 32, at least 85%amino acid identity with the amino acid sequence of SEQ ID NO: 32, atleast 90% amino acid identity with the amino acid sequence of SEQ ID NO:32 or at least 95% amino acid identity with the amino acid sequence ofSEQ ID NO: 32. In yet other aspects of this embodiment, a VEGF comprisesa polypeptide having, e.g., at most 70% amino acid identity with theamino acid sequence of SEQ ID NO: 32, at most 75% amino acid identitywith the amino acid sequence of SEQ ID NO: 32, at most 80% amino acididentity with the amino acid sequence of SEQ ID NO: 32, at most 85%amino acid identity with the amino acid sequence of SEQ ID NO: 32, atmost 90% amino acid identity with the amino acid sequence of SEQ ID NO:32 or at most 95% amino acid identity with the amino acid sequence ofSEQ ID NO: 32.

In other aspects of this embodiment, a VEGF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to theamino acid sequence of SEQ ID NO: 32. In other aspects of thisembodiment, a VEGF comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to the amino acid sequence of SEQ IDNO: 32. In yet other aspects of this embodiment, a VEGF comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to the amino acid sequence of SEQ ID NO: 32. In other aspectsof this embodiment, a VEGF comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to the amino acid sequenceof SEQ ID NO: 32. In still other aspects of this embodiment, a VEGFcomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to the amino acid sequence of SEQ ID NO: 32. In otheraspects of this embodiment, a VEGF comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to the amino acid sequenceof SEQ ID NO: 32.

In other aspects of this embodiment, a VEGF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to the aminoacid sequence of SEQ ID NO: 32. In other aspects of this embodiment, aVEGF comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to the amino acid sequence of SEQ ID NO: 32. Inyet other aspects of this embodiment, a VEGF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to the aminoacid sequence of SEQ ID NO: 32. In other aspects of this embodiment, aVEGF comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to the amino acid sequence of SEQ ID NO: 32. In stillother aspects of this embodiment, a VEGF comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to the amino acidsequence of SEQ ID NO: 32. In other aspects of this embodiment, a VEGFcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to the amino acid sequence of SEQ ID NO: 32.

In another embodiment, an enhanced binding domain derived from an IGF-1.In another embodiment, an enhanced binding domain derived from an IGF-1of SEQ ID NO: 33. In an aspect of this embodiment, an enhanced bindingdomain derived from an IGF-1 comprises amino acids 52-109 or amino acids49-118 of SEQ ID NO: 33.

In other aspects of this embodiment, an IGF-1 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 52-109or amino acids 49-118 of SEQ ID NO: 33, at least 75% amino acid identitywith amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 33, at least80% amino acid identity with amino acids 52-109 or amino acids 49-118 ofSEQ ID NO: 33, at least 85% amino acid identity with amino acids 52-109or amino acids 49-118 of SEQ ID NO: 33, at least 90% amino acid identitywith amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 33 or atleast 95% amino acid identity with amino acids 52-109 or amino acids49-118 of SEQ ID NO: 33. In yet other aspects of this embodiment, anIGF-1 comprises a polypeptide having, e.g., at most 70% amino acididentity with amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 33,at most 75% amino acid identity with amino acids 52-109 or amino acids49-118 of SEQ ID NO: 33, at most 80% amino acid identity with aminoacids 52-109 or amino acids 49-118 of SEQ ID NO: 33, at most 85% aminoacid identity with amino acids 52-109 or amino acids 49-118 of SEQ IDNO: 33, at most 90% amino acid identity with amino acids 52-109 or aminoacids 49-118 of SEQ ID NO: 33 or at most 95% amino acid identity withamino acids 52-109 or amino acids 49-118 of SEQ ID NO: 33.

In other aspects of this embodiment, an IGF-1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 52-109 or amino acids 49-118 of SEQ ID NO: 33. In other aspects ofthis embodiment, an IGF-1 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 52-109or amino acids 49-118 of SEQ ID NO: 33. In yet other aspects of thisembodiment, an IGF-1 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 52-109 or amino acids49-118 of SEQ ID NO: 33. In other aspects of this embodiment, an IGF-1comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 52-109 or amino acids 49-118 of SEQ IDNO: 33. In still other aspects of this embodiment, an IGF-1 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 33.In other aspects of this embodiment, an IGF-1 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 52-109 or amino acids 49-118 of SEQ ID NO: 33.

In other aspects of this embodiment, an IGF-1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 52-109 or amino acids 49-118 of SEQ ID NO: 33. In other aspects ofthis embodiment, an IGF-1 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 52-109 oramino acids 49-118 of SEQ ID NO: 33. In yet other aspects of thisembodiment, an IGF-1 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 52-109 or amino acids49-118 of SEQ ID NO: 33. In other aspects of this embodiment, an IGF-1comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 33.In still other aspects of this embodiment, an IGF-1 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 52-109 or amino acids 49-118 of SEQ ID NO: 33. In otheraspects of this embodiment, an IGF-1 comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to amino acids 52-109 oramino acids 49-118 of SEQ ID NO: 33.

In another embodiment, an enhanced binding domain derived from an IGF-2.In another embodiment, an enhanced binding domain derived from an IGF-2of SEQ ID NO: 34. In an aspect of this embodiment, an enhanced bindingdomain derived from an IGF-2 comprises amino acids 31-84 or amino acids25-180 of SEQ ID NO: 34.

In other aspects of this embodiment, an IGF-2 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 31-84 oramino acids 25-180 of SEQ ID NO: 34, at least 75% amino acid identitywith amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 34, at least80% amino acid identity with amino acids 31-84 or amino acids 25-180 ofSEQ ID NO: 34, at least 85% amino acid identity with amino acids 31-84or amino acids 25-180 of SEQ ID NO: 34, at least 90% amino acid identitywith amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 34 or atleast 95% amino acid identity with amino acids 31-84 or amino acids25-180 of SEQ ID NO: 34. In yet other aspects of this embodiment, anIGF-2 comprises a polypeptide having, e.g., at most 70% amino acididentity with amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 34,at most 75% amino acid identity with amino acids 31-84 or amino acids25-180 of SEQ ID NO: 34, at most 80% amino acid identity with aminoacids 31-84 or amino acids 25-180 of SEQ ID NO: 34, at most 85% aminoacid identity with amino acids 31-84 or amino acids 25-180 of SEQ ID NO:34, at most 90% amino acid identity with amino acids 31-84 or aminoacids 25-180 of SEQ ID NO: 34 or at most 95% amino acid identity withamino acids 31-84 or amino acids 25-180 of SEQ ID NO: 34.

In other aspects of this embodiment, an IGF-2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 31-84 or amino acids 25-180 of SEQ ID NO: 34. In other aspects ofthis embodiment, an IGF-2 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 31-84 oramino acids 25-180 of SEQ ID NO: 34. In yet other aspects of thisembodiment, an IGF-2 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 31-84 or amino acids 25-180of SEQ ID NO: 34. In other aspects of this embodiment, an IGF-2comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 31-84 or amino acids 25-180 of SEQ IDNO: 34. In still other aspects of this embodiment, an IGF-2 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 34. Inother aspects of this embodiment, an IGF-2 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 31-84 or amino acids 25-180 of SEQ ID NO: 34.

In other aspects of this embodiment, an IGF-2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 31-84 or amino acids 25-180 of SEQ ID NO: 34. In other aspects ofthis embodiment, an IGF-2 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 31-84 oramino acids 25-180 of SEQ ID NO: 34. In yet other aspects of thisembodiment, an IGF-2 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 31-84 or amino acids 25-180of SEQ ID NO: 34. In other aspects of this embodiment, an IGF-2comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 34. Instill other aspects of this embodiment, an IGF-2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids31-84 or amino acids 25-180 of SEQ ID NO: 34. In other aspects of thisembodiment, an IGF-2 comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 31-84 or amino acids 25-180of SEQ ID NO: 34.

In another embodiment, an enhanced binding domain derived from an EGF.In another embodiment, an enhanced binding domain derived from an EGF ofSEQ ID NO: 35. In aspects of this embodiment, an EGF comprises apolypeptide having, e.g., at least 70% amino acid identity with theamino acid sequence of SEQ ID NO: 35, at least 75% amino acid identitywith the amino acid sequence of SEQ ID NO: 35, at least 80% amino acididentity with the amino acid sequence of SEQ ID NO: 35, at least 85%amino acid identity with the amino acid sequence of SEQ ID NO: 35, atleast 90% amino acid identity with the amino acid sequence of SEQ ID NO:35 or at least 95% amino acid identity with the amino acid sequence ofSEQ ID NO: 35. In yet other aspects of this embodiment, an EGF comprisesa polypeptide having, e.g., at most 70% amino acid identity with theamino acid sequence of SEQ ID NO: 35, at most 75% amino acid identitywith the amino acid sequence of SEQ ID NO: 35, at most 80% amino acididentity with the amino acid sequence of SEQ ID NO: 35, at most 85%amino acid identity with the amino acid sequence of SEQ ID NO: 35, atmost 90% amino acid identity with the amino acid sequence of SEQ ID NO:35 or at most 95% amino acid identity with the amino acid sequence ofSEQ ID NO: 35.

In other aspects of this embodiment, an EGF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to theamino acid sequence of SEQ ID NO: 35. In other aspects of thisembodiment, an EGF comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to the amino acid sequence of SEQ IDNO: 35. In yet other aspects of this embodiment, an EGF comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to the amino acid sequence of SEQ ID NO: 35. In other aspectsof this embodiment, an EGF comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to the amino acid sequenceof SEQ ID NO: 35. In still other aspects of this embodiment, an EGFcomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to the amino acid sequence of SEQ ID NO: 35. In otheraspects of this embodiment, an EGF comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to the amino acid sequenceof SEQ ID NO: 35.

In other aspects of this embodiment, an EGF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to the aminoacid sequence of SEQ ID NO: 35. In other aspects of this embodiment, anEGF comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to the amino acid sequence of SEQ ID NO: 35. Inyet other aspects of this embodiment, an EGF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to the aminoacid sequence of SEQ ID NO: 35. In other aspects of this embodiment, anEGF comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to the amino acid sequence of SEQ ID NO: 35. In stillother aspects of this embodiment, an EGF comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to the amino acidsequence of SEQ ID NO: 35. In other aspects of this embodiment, an EGFcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to the amino acid sequence of SEQ ID NO: 35.

Another example of an enhanced targeting domain disclosed in the presentspecification is, e.g., a neurotrophin, such as, e.g., a NGF, a BDNF, aNT-3 or a NT-5.

Thus, in an embodiment, an enhanced binding domain derived from a NGF.In another embodiment, an enhanced binding domain derived from a NGF ofSEQ ID NO: 36. In an aspect of this embodiment, an enhanced bindingdomain derived from a NGF comprises amino acids 139-257 of SEQ ID NO:36.

In other aspects of this embodiment, a NGF comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 139-257of SEQ ID NO: 36, at least 75% amino acid identity with amino acids139-257 of SEQ ID NO: 36, at least 80% amino acid identity with aminoacids 139-257 of SEQ ID NO: 36, at least 85% amino acid identity withamino acids 139-257 of SEQ ID NO: 36, at least 90% amino acid identitywith amino acids 139-257 of SEQ ID NO: 36 or at least 95% amino acididentity with amino acids 139-257 of SEQ ID NO: 36. In yet other aspectsof this embodiment, a NGF comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 139-257 of SEQ ID NO: 36, atmost 75% amino acid identity with amino acids 139-257 of SEQ ID NO: 36,at most 80% amino acid identity with amino acids 139-257 of SEQ ID NO:36, at most 85% amino acid identity with amino acids 139-257 of SEQ IDNO: 36, at most 90% amino acid identity with amino acids 139-257 of SEQID NO: 36 or at most 95% amino acid identity with amino acids 139-257 ofSEQ ID NO: 36.

In other aspects of this embodiment, a NGF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 139-257 of SEQ ID NO: 36. In other aspects of this embodiment, aNGF comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 139-257 of SEQ ID NO: 36. In yetother aspects of this embodiment, a NGF comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids139-257 of SEQ ID NO: 36. In other aspects of this embodiment, a NGFcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 139-257 of SEQ ID NO: 36. In stillother aspects of this embodiment, a NGF comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids139-257 of SEQ ID NO: 36. In other aspects of this embodiment, a NGFcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 139-257 of SEQ ID NO: 36.

In other aspects of this embodiment, a NGF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 139-257 of SEQ ID NO: 36. In other aspects of this embodiment, aNGF comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 139-257 of SEQ ID NO: 36. In yetother aspects of this embodiment, a NGF comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 139-257 ofSEQ ID NO: 36. In other aspects of this embodiment, a NGF comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 139-257 of SEQ ID NO: 36. In still other aspects of thisembodiment, a NGF comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 139-257 of SEQ ID NO: 36.In other aspects of this embodiment, a NGF comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids139-257 of SEQ ID NO: 36.

In another embodiment, an enhanced binding domain derived from a BDGF.In another embodiment, an enhanced binding domain derived from a BDGF ofSEQ ID NO: 37. In an aspect of this embodiment, an enhanced bindingdomain derived from a BDGF comprises amino acids 133-240 or amino acids129-247 of SEQ ID NO: 37.

In other aspects of this embodiment, a BDGF comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 133-240or amino acids 129-247 of SEQ ID NO: 37, at least 75% amino acididentity with amino acids 133-240 or amino acids 129-247 of SEQ ID NO:37, at least 80% amino acid identity with amino acids 133-240 or aminoacids 129-247 of SEQ ID NO: 37, at least 85% amino acid identity withamino acids 133-240 or amino acids 129-247 of SEQ ID NO: 37, at least90% amino acid identity with amino acids 133-240 or amino acids 129-247of SEQ ID NO: 37 or at least 95% amino acid identity with amino acids133-240 or amino acids 129-247 of SEQ ID NO: 37. In yet other aspects ofthis embodiment, a BDGF comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 133-240 or amino acids 129-247of SEQ ID NO: 37, at most 75% amino acid identity with amino acids133-240 or amino acids 129-247 of SEQ ID NO: 37, at most 80% amino acididentity with amino acids 133-240 or amino acids 129-247 of SEQ ID NO:37, at most 85% amino acid identity with amino acids 133-240 or aminoacids 129-247 of SEQ ID NO: 37, at most 90% amino acid identity withamino acids 133-240 or amino acids 129-247 of SEQ ID NO: 37 or at most95% amino acid identity with amino acids 133-240 or amino acids 129-247of SEQ ID NO: 37.

In other aspects of this embodiment, a BDGF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 133-240 or amino acids 129-247 of SEQ ID NO: 37. In other aspectsof this embodiment, a BDGF comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 133-240or amino acids 129-247 of SEQ ID NO: 37. In yet other aspects of thisembodiment, a BDGF comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 133-240 or amino acids129-247 of SEQ ID NO: 37. In other aspects of this embodiment, a BDGFcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 133-240 or amino acids 129-247 of SEQID NO: 37. In still other aspects of this embodiment, a BDGF comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 37.In other aspects of this embodiment, a BDGF comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 133-240 or amino acids 129-247 of SEQ ID NO: 37.

In other aspects of this embodiment, a BDGF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 133-240 or amino acids 129-247 of SEQ ID NO: 37. In other aspectsof this embodiment, a BDGF comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 133-240 oramino acids 129-247 of SEQ ID NO: 37. In yet other aspects of thisembodiment, a BDGF comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 133-240 or amino acids129-247 of SEQ ID NO: 37. In other aspects of this embodiment, a BDGFcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 37.In still other aspects of this embodiment, a BDGF comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 133-240 or amino acids 129-247 of SEQ ID NO: 37. In otheraspects of this embodiment, a BDGF comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 133-240 or aminoacids 129-247 of SEQ ID NO: 37.

In another embodiment, an enhanced binding domain derived from a NT-3.In another embodiment, an enhanced binding domain derived from a NT-3 ofSEQ ID NO: 38. In an aspect of this embodiment, an enhanced bindingdomain derived from a NT-3 comprises amino acids 144-249 or amino acids19-257 of SEQ ID NO: 38.

In other aspects of this embodiment, a NT-3 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 144-249or amino acids 19-257 of SEQ ID NO: 38, at least 75% amino acid identitywith amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 38, atleast 80% amino acid identity with amino acids 144-249 or amino acids19-257 of SEQ ID NO: 38, at least 85% amino acid identity with aminoacids 144-249 or amino acids 19-257 of SEQ ID NO: 38, at least 90% aminoacid identity with amino acids 144-249 or amino acids 19-257 of SEQ IDNO: 38 or at least 95% amino acid identity with amino acids 144-249 oramino acids 19-257 of SEQ ID NO: 38. In yet other aspects of thisembodiment, a NT-3 comprises a polypeptide having, e.g., at most 70%amino acid identity with amino acids 144-249 or amino acids 19-257 ofSEQ ID NO: 38, at most 75% amino acid identity with amino acids 144-249or amino acids 19-257 of SEQ ID NO: 38, at most 80% amino acid identitywith amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 38, at most85% amino acid identity with amino acids 144-249 or amino acids 19-257of SEQ ID NO: 38, at most 90% amino acid identity with amino acids144-249 or amino acids 19-257 of SEQ ID NO: 38 or at most 95% amino acididentity with amino acids 144-249 or amino acids 19-257 of SEQ ID NO:38.

In other aspects of this embodiment, a NT-3 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 144-249 or amino acids 19-257 of SEQ ID NO: 38. In other aspectsof this embodiment, a NT-3 comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 144-249or amino acids 19-257 of SEQ ID NO: 38. In yet other aspects of thisembodiment, a NT-3 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 144-249 or amino acids19-257 of SEQ ID NO: 38. In other aspects of this embodiment, a NT-3comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 144-249 or amino acids 19-257 of SEQID NO: 38. In still other aspects of this embodiment, a NT-3 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 38.In other aspects of this embodiment, a NT-3 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 144-249 or amino acids 19-257 of SEQ ID NO: 38.

In other aspects of this embodiment, a NT-3 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 144-249 or amino acids 19-257 of SEQ ID NO: 38. In other aspectsof this embodiment, a NT-3 comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 144-249 oramino acids 19-257 of SEQ ID NO: 38. In yet other aspects of thisembodiment, a NT-3 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 144-249 or amino acids19-257 of SEQ ID NO: 38. In other aspects of this embodiment, a NT-3comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 38.In still other aspects of this embodiment, a NT-3 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 144-249 or amino acids 19-257 of SEQ ID NO: 38. In otheraspects of this embodiment, a NT-3 comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 144-249 or aminoacids 19-257 of SEQ ID NO: 38.

In another embodiment, an enhanced binding domain derived from a NT-4/5.In another embodiment, an enhanced binding domain derived from a NT-4/5of SEQ ID NO: 39. In an aspect of this embodiment, an enhanced bindingdomain derived from a NT-4/5 comprises amino acids 89-202 or amino acids81-210 of SEQ ID NO: 39.

In other aspects of this embodiment, a NT-4/5 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 89-202or amino acids 81-210 of SEQ ID NO: 39, at least 75% amino acid identitywith amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 39, at least80% amino acid identity with amino acids 89-202 or amino acids 81-210 ofSEQ ID NO: 39, at least 85% amino acid identity with amino acids 89-202or amino acids 81-210 of SEQ ID NO: 39, at least 90% amino acid identitywith amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 39 or atleast 95% amino acid identity with amino acids 89-202 or amino acids81-210 of SEQ ID NO: 39. In yet other aspects of this embodiment, aNT-4/5 comprises a polypeptide having, e.g., at most 70% amino acididentity with amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 39,at most 75% amino acid identity with amino acids 89-202 or amino acids81-210 of SEQ ID NO: 39, at most 80% amino acid identity with aminoacids 89-202 or amino acids 81-210 of SEQ ID NO: 39, at most 85% aminoacid identity with amino acids 89-202 or amino acids 81-210 of SEQ IDNO: 39, at most 90% amino acid identity with amino acids 89-202 or aminoacids 81-210 of SEQ ID NO: 39 or at most 95% amino acid identity withamino acids 89-202 or amino acids 81-210 of SEQ ID NO: 39.

In other aspects of this embodiment, a NT-4/5 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 89-202 or amino acids 81-210 of SEQ ID NO: 39. In other aspects ofthis embodiment, a NT-4/5 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 89-202or amino acids 81-210 of SEQ ID NO: 39. In yet other aspects of thisembodiment, a NT-4/5 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 89-202 or amino acids81-210 of SEQ ID NO: 39. In other aspects of this embodiment, a NT-4/5comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 89-202 or amino acids 81-210 of SEQ IDNO: 39. In still other aspects of this embodiment, a NT-4/5 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 39.In other aspects of this embodiment, a NT-4/5 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 89-202 or amino acids 81-210 of SEQ ID NO: 39.

In other aspects of this embodiment, a NT-4/5 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 89-202 or amino acids 81-210 of SEQ ID NO: 39. In other aspects ofthis embodiment, a NT-4/5 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 89-202 oramino acids 81-210 of SEQ ID NO: 39. In yet other aspects of thisembodiment, a NT-4/5 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 89-202 or amino acids81-210 of SEQ ID NO: 39. In other aspects of this embodiment, a NT-4/5comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 39.In still other aspects of this embodiment, a NT-4/5 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 89-202 or amino acids 81-210 of SEQ ID NO: 39. In otheraspects of this embodiment, a NT-4/5 comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to amino acids 89-202 oramino acids 81-210 of SEQ ID NO: 39.

Another example of an enhanced targeting domain disclosed in the presentspecification is, e.g., a GDNF, a neurturin, a persephrin or an artemin.

Thus, in an embodiment, an enhanced binding domain derived from a GDNF.In another embodiment, an enhanced binding domain derived from a GDNF ofSEQ ID NO: 40. In an aspect of this embodiment, an enhanced bindingdomain derived from a GDNF comprises amino acids 118-211 of SEQ ID NO:40.

In other aspects of this embodiment, a GDNF comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 118-211of SEQ ID NO: 40, at least 75% amino acid identity with amino acids118-211 of SEQ ID NO: 40, at least 80% amino acid identity with aminoacids 118-211 of SEQ ID NO: 40, at least 85% amino acid identity withamino acids 118-211 of SEQ ID NO: 40, at least 90% amino acid identitywith amino acids 118-211 of SEQ ID NO: 40 or at least 95% amino acididentity with amino acids 118-211 of SEQ ID NO: 40. In yet other aspectsof this embodiment, a GDNF comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 118-211 of SEQ ID NO: 40, atmost 75% amino acid identity with amino acids 118-211 of SEQ ID NO: 40,at most 80% amino acid identity with amino acids 118-211 of SEQ ID NO:40, at most 85% amino acid identity with amino acids 118-211 of SEQ IDNO: 40, at most 90% amino acid identity with amino acids 118-211 of SEQID NO: 40 or at most 95% amino acid identity with amino acids 118-211 ofSEQ ID NO: 40.

In other aspects of this embodiment, a GDNF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 118-211 of SEQ ID NO: 40. In other aspects of this embodiment, aGDNF comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 118-211 of SEQ ID NO: 40. In yetother aspects of this embodiment, a GDNF comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids118-211 of SEQ ID NO: 40. In other aspects of this embodiment, a GDNFcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 118-211 of SEQ ID NO: 40. In stillother aspects of this embodiment, a GDNF comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids118-211 of SEQ ID NO: 40. In other aspects of this embodiment, a GDNFcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 118-211 of SEQ ID NO: 40.

In other aspects of this embodiment, a GDNF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 118-211 of SEQ ID NO: 40. In other aspects of this embodiment, aGDNF comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 118-211 of SEQ ID NO: 40. In yetother aspects of this embodiment, a GDNF comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 118-211 ofSEQ ID NO: 40. In other aspects of this embodiment, a GDNF comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 118-211 of SEQ ID NO: 40. In still other aspects of thisembodiment, a GDNF comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 118-211 of SEQ ID NO: 40.In other aspects of this embodiment, a GDNF comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids118-211 of SEQ ID NO: 40.

In another embodiment, an enhanced binding domain derived from aNeurturin. In another embodiment, an enhanced binding domain derivedfrom a Neurturin of SEQ ID NO: 41. In an aspect of this embodiment, anenhanced binding domain derived from a Neurturin comprises amino acids107-196 or amino acids 96-197 of SEQ ID NO: 41.

In other aspects of this embodiment, a Neurturin comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 107-196or amino acids 96-197 of SEQ ID NO: 41, at least 75% amino acid identitywith amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 41, atleast 80% amino acid identity with amino acids 107-196 or amino acids96-197 of SEQ ID NO: 41, at least 85% amino acid identity with aminoacids 107-196 or amino acids 96-197 of SEQ ID NO: 41, at least 90% aminoacid identity with amino acids 107-196 or amino acids 96-197 of SEQ IDNO: 41 or at least 95% amino acid identity with amino acids 107-196 oramino acids 96-197 of SEQ ID NO: 41. In yet other aspects of thisembodiment, a Neurturin comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 107-196 or amino acids 96-197of SEQ ID NO: 41, at most 75% amino acid identity with amino acids107-196 or amino acids 96-197 of SEQ ID NO: 41, at most 80% amino acididentity with amino acids 107-196 or amino acids 96-197 of SEQ ID NO:41, at most 85% amino acid identity with amino acids 107-196 or aminoacids 96-197 of SEQ ID NO: 41, at most 90% amino acid identity withamino acids 107-196 or amino acids 96-197 of SEQ ID NO: 41 or at most95% amino acid identity with amino acids 107-196 or amino acids 96-197of SEQ ID NO: 41.

In other aspects of this embodiment, a Neurturin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 107-196 or amino acids 96-197 of SEQ ID NO: 41. In other aspectsof this embodiment, a Neurturin comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 107-196or amino acids 96-197 of SEQ ID NO: 41. In yet other aspects of thisembodiment, a Neurturin comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 107-196 oramino acids 96-197 of SEQ ID NO: 41. In other aspects of thisembodiment, a Neurturin comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 107-196 oramino acids 96-197 of SEQ ID NO: 41. In still other aspects of thisembodiment, a Neurturin comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 107-196 oramino acids 96-197 of SEQ ID NO: 41. In other aspects of thisembodiment, a Neurturin comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 107-196 oramino acids 96-197 of SEQ ID NO: 41.

In other aspects of this embodiment, a Neurturin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 107-196 or amino acids 96-197 of SEQ ID NO: 41. In other aspectsof this embodiment, a Neurturin comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 107-196 oramino acids 96-197 of SEQ ID NO: 41. In yet other aspects of thisembodiment, a Neurturin comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 107-196 or aminoacids 96-197 of SEQ ID NO: 41. In other aspects of this embodiment, aNeurturin comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid deletions relative to amino acids 107-196 or amino acids 96-197 ofSEQ ID NO: 41. In still other aspects of this embodiment, a Neurturincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 41.In other aspects of this embodiment, a Neurturin comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids107-196 or amino acids 96-197 of SEQ ID NO: 41.

In another embodiment, an enhanced binding domain derived from aPersephrin. In another embodiment, an enhanced binding domain derivedfrom a Persephrin of SEQ ID NO: 42. In an aspect of this embodiment, anenhanced binding domain derived from a Persephrin comprises amino acids66-155 of SEQ ID NO: 42.

In other aspects of this embodiment, a Persephrin comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 66-155 of SEQ ID NO: 42, at least 75% amino acid identity withamino acids 66-155 of SEQ ID NO: 42, at least 80% amino acid identitywith amino acids 66-155 of SEQ ID NO: 42, at least 85% amino acididentity with amino acids 66-155 of SEQ ID NO: 42, at least 90% aminoacid identity with amino acids 66-155 of SEQ ID NO: 42 or at least 95%amino acid identity with amino acids 66-155 of SEQ ID NO: 42. In yetother aspects of this embodiment, a Persephrin comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 66-155 ofSEQ ID NO: 42, at most 75% amino acid identity with amino acids 66-155of SEQ ID NO: 42, at most 80% amino acid identity with amino acids66-155 of SEQ ID NO: 42, at most 85% amino acid identity with aminoacids 66-155 of SEQ ID NO: 42, at most 90% amino acid identity withamino acids 66-155 of SEQ ID NO: 42 or at most 95% amino acid identitywith amino acids 66-155 of SEQ ID NO: 42.

In other aspects of this embodiment, a Persephrin comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid substitutionsrelative to amino acids 66-155 of SEQ ID NO: 42. In other aspects ofthis embodiment, a Persephrin comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 66-155of SEQ ID NO: 42. In yet other aspects of this embodiment, a Persephrincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 66-155 of SEQ ID NO: 42. In otheraspects of this embodiment, a Persephrin comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids 66-155of SEQ ID NO: 42. In still other aspects of this embodiment, aPersephrin comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid additions relative to amino acids 66-155 of SEQ ID NO: 42. Inother aspects of this embodiment, a Persephrin comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 66-155 of SEQ ID NO: 42.

In other aspects of this embodiment, a Persephrin comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to amino acids 66-155 of SEQ ID NO: 42. In other aspects ofthis embodiment, a Persephrin comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 66-155 ofSEQ ID NO: 42. In yet other aspects of this embodiment, a Persephrincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 66-155 of SEQ ID NO: 42. In other aspects ofthis embodiment, a Persephrin comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 66-155 of SEQ IDNO: 42. In still other aspects of this embodiment, a Persephrincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 66-155 of SEQ ID NO: 42. In other aspects ofthis embodiment, a Persephrin comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 66-155 of SEQ IDNO: 42.

In another embodiment, an enhanced binding domain derived from anArtemin. In another embodiment, an enhanced binding domain derived froman Artemin of SEQ ID NO: 43. In an aspect of this embodiment, anenhanced binding domain derived from an Artemin comprises amino acids123-218 of SEQ ID NO: 43.

In other aspects of this embodiment, an Artemin comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 123-218of SEQ ID NO: 43, at least 75% amino acid identity with amino acids123-218 of SEQ ID NO: 43, at least 80% amino acid identity with aminoacids 123-218 of SEQ ID NO: 43, at least 85% amino acid identity withamino acids 123-218 of SEQ ID NO: 43, at least 90% amino acid identitywith amino acids 123-218 of SEQ ID NO: 43 or at least 95% amino acididentity with amino acids 123-218 of SEQ ID NO: 43. In yet other aspectsof this embodiment, an Artemin comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 123-218 of SEQ ID NO: 43,at most 75% amino acid identity with amino acids 123-218 of SEQ ID NO:43, at most 80% amino acid identity with amino acids 123-218 of SEQ IDNO: 43, at most 85% amino acid identity with amino acids 123-218 of SEQID NO: 43, at most 90% amino acid identity with amino acids 123-218 ofSEQ ID NO: 43 or at most 95% amino acid identity with amino acids123-218 of SEQ ID NO: 43.

In other aspects of this embodiment, an Artemin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 123-218 of SEQ ID NO: 43. In other aspects of this embodiment, anArtemin comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 123-218 of SEQ ID NO: 43. In yetother aspects of this embodiment, an Artemin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 123-218 of SEQ ID NO: 43. In other aspects of this embodiment, anArtemin comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 123-218 of SEQ ID NO: 43. In stillother aspects of this embodiment, an Artemin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 123-218 of SEQ ID NO: 43. In other aspects of this embodiment, anArtemin comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 123-218 of SEQ ID NO: 43.

In other aspects of this embodiment, an Artemin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 123-218 of SEQ ID NO: 43. In other aspects of this embodiment, anArtemin comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 123-218 of SEQ ID NO: 43. In yetother aspects of this embodiment, an Artemin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids123-218 of SEQ ID NO: 43. In other aspects of this embodiment, anArtemin comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to amino acids 123-218 of SEQ ID NO: 43. In stillother aspects of this embodiment, an Artemin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids123-218 of SEQ ID NO: 43. In other aspects of this embodiment, anArtemin comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 123-218 of SEQ ID NO: 43.

Another example of an enhanced targeting domain disclosed in the presentspecification is, e.g., a TGFβs, such as, e.g., TGFβ1, TGFβ2, TGFβ3 orTGFβ4.

Thus, in an embodiment, an enhanced binding domain derived from a TGFβ1.In another embodiment, an enhanced binding domain derived from a TGFβ1of SEQ ID NO: 44. In an aspect of this embodiment, an enhanced bindingdomain derived from a TGFβ1 comprises amino acids 293-390 of SEQ ID NO:44.

In other aspects of this embodiment, a TGFβ1 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 293-390of SEQ ID NO: 44, at least 75% amino acid identity with amino acids293-390 of SEQ ID NO: 44, at least 80% amino acid identity with aminoacids 293-390 of SEQ ID NO: 44, at least 85% amino acid identity withamino acids 293-390 of SEQ ID NO: 44, at least 90% amino acid identitywith amino acids 293-390 of SEQ ID NO: 44 or at least 95% amino acididentity with amino acids 293-390 of SEQ ID NO: 44. In yet other aspectsof this embodiment, a TGFβ1 comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 293-390 of SEQ ID NO: 44,at most 75% amino acid identity with amino acids 293-390 of SEQ ID NO:44, at most 80% amino acid identity with amino acids 293-390 of SEQ IDNO: 44, at most 85% amino acid identity with amino acids 293-390 of SEQID NO: 44, at most 90% amino acid identity with amino acids 293-390 ofSEQ ID NO: 44 or at most 95% amino acid identity with amino acids293-390 of SEQ ID NO: 44.

In other aspects of this embodiment, a TGFβ1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 293-390 of SEQ ID NO: 44. In other aspects of this embodiment, aTGFβ1 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 293-390 of SEQ ID NO: 44. In yetother aspects of this embodiment, a TGFβ1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 293-390 of SEQ ID NO: 44. In other aspects of this embodiment, aTGFβ1 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 293-390 of SEQ ID NO: 44. In stillother aspects of this embodiment, a TGFβ1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 293-390 of SEQ ID NO: 44. In other aspects of this embodiment, aTGFβ1 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 293-390 of SEQ ID NO: 44.

In other aspects of this embodiment, a TGFβ1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 293-390 of SEQ ID NO: 44. In other aspects of this embodiment, aTGFβ1 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 293-390 of SEQ ID NO: 44. In yetother aspects of this embodiment, a TGFβ1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids293-390 of SEQ ID NO: 44. In other aspects of this embodiment, a TGFβ1comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 293-390 of SEQ ID NO: 44. In still other aspectsof this embodiment, a TGFβ1 comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 293-390 of SEQID NO: 44. In other aspects of this embodiment, a TGFβ1 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 293-390 of SEQ ID NO: 44.

In another embodiment, an enhanced binding domain derived from a TGFβ2.In another embodiment, an enhanced binding domain derived from a TGFβ2of SEQ ID NO: 44. In an aspect of this embodiment, an enhanced bindingdomain derived from a TGFβ2 comprises amino acids 317-414 of SEQ ID NO:45.

In other aspects of this embodiment, a TGFβ2 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 317-414of SEQ ID NO: 45, at least 75% amino acid identity with amino acids317-414 of SEQ ID NO: 45, at least 80% amino acid identity with aminoacids 317-414 of SEQ ID NO: 45, at least 85% amino acid identity withamino acids 317-414 of SEQ ID NO: 45, at least 90% amino acid identitywith amino acids 317-414 of SEQ ID NO: 45 or at least 95% amino acididentity with amino acids 317-414 of SEQ ID NO: 45. In yet other aspectsof this embodiment, a TGFβ2 comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 317-414 of SEQ ID NO: 45,at most 75% amino acid identity with amino acids 317-414 of SEQ ID NO:45, at most 80% amino acid identity with amino acids 317-414 of SEQ IDNO: 45, at most 85% amino acid identity with amino acids 317-414 of SEQID NO: 45, at most 90% amino acid identity with amino acids 317-414 ofSEQ ID NO: 45 or at most 95% amino acid identity with amino acids317-414 of SEQ ID NO: 45.

In other aspects of this embodiment, a TGFβ2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 317-414 of SEQ ID NO: 45. In other aspects of this embodiment, aTGFβ2 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 317-414 of SEQ ID NO: 45. In yetother aspects of this embodiment, a TGFβ2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 317-414 of SEQ ID NO: 45. In other aspects of this embodiment, aTGFβ2 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 317-414 of SEQ ID NO: 45. In stillother aspects of this embodiment, a TGFβ2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 317-414 of SEQ ID NO: 45. In other aspects of this embodiment, aTGFβ2 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 317-414 of SEQ ID NO: 45.

In other aspects of this embodiment, a TGFβ2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 317-414 of SEQ ID NO: 45. In other aspects of this embodiment, aTGFβ2 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 317-414 of SEQ ID NO: 45. In yetother aspects of this embodiment, a TGFβ2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids317-414 of SEQ ID NO: 45. In other aspects of this embodiment, a TGFβ2comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 317-414 of SEQ ID NO: 45. In still other aspectsof this embodiment, a TGFβ2 comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 317-414 of SEQID NO: 45. In other aspects of this embodiment, a TGFβ2 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 317-414 of SEQ ID NO: 45.

In another embodiment, an enhanced binding domain derived from a TGFβ3.In another embodiment, an enhanced binding domain derived from a TGFβ3of SEQ ID NO: 44. In an aspect of this embodiment, an enhanced bindingdomain derived from a TGFβ3 comprises amino acids 315-412 of SEQ ID NO:46.

In other aspects of this embodiment, a TGFβ3 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 315-412of SEQ ID NO: 46, at least 75% amino acid identity with amino acids315-412 of SEQ ID NO: 46, at least 80% amino acid identity with aminoacids 315-412 of SEQ ID NO: 46, at least 85% amino acid identity withamino acids 315-412 of SEQ ID NO: 46, at least 90% amino acid identitywith amino acids 315-412 of SEQ ID NO: 46 or at least 95% amino acididentity with amino acids 315-412 of SEQ ID NO: 46. In yet other aspectsof this embodiment, a TGFβ3 comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 315-412 of SEQ ID NO: 46,at most 75% amino acid identity with amino acids 315-412 of SEQ ID NO:46, at most 80% amino acid identity with amino acids 315-412 of SEQ IDNO: 46, at most 85% amino acid identity with amino acids 315-412 of SEQID NO: 46, at most 90% amino acid identity with amino acids 315-412 ofSEQ ID NO: 46 or at most 95% amino acid identity with amino acids315-412 of SEQ ID NO: 46.

In other aspects of this embodiment, a TGFβ3 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 315-412 of SEQ ID NO: 46. In other aspects of this embodiment, aTGFβ3 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 315-412 of SEQ ID NO: 46. In yetother aspects of this embodiment, a TGFβ3 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 315-412 of SEQ ID NO: 46. In other aspects of this embodiment, aTGFβ3 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 315-412 of SEQ ID NO: 46. In stillother aspects of this embodiment, a TGFβ3 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 315-412 of SEQ ID NO: 46. In other aspects of this embodiment, aTGFβ3 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 315-412 of SEQ ID NO: 46.

In other aspects of this embodiment, a TGFβ3 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 315-412 of SEQ ID NO: 46. In other aspects of this embodiment, aTGFβ3 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 315-412 of SEQ ID NO: 46. In yetother aspects of this embodiment, a TGFβ3 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids315-412 of SEQ ID NO: 46. In other aspects of this embodiment, a TGFβ3comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 315-412 of SEQ ID NO: 46. In still other aspectsof this embodiment, a TGFβ3 comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 315-412 of SEQID NO: 46. In other aspects of this embodiment, a TGFβ3 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 315-412 of SEQ ID NO: 46.

In another embodiment, an enhanced binding domain derived from a TGFβ4.In another embodiment, an enhanced binding domain derived from a TGFβ4of SEQ ID NO: 44. In an aspect of this embodiment, an enhanced bindingdomain derived from a TGFβ4 comprises amino acids 276-373 of SEQ ID NO:47.

In other aspects of this embodiment, a TGFβ4 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 276-373of SEQ ID NO: 47, at least 75% amino acid identity with amino acids276-373 of SEQ ID NO: 47, at least 80% amino acid identity with aminoacids 276-373 of SEQ ID NO: 47, at least 85% amino acid identity withamino acids 276-373 of SEQ ID NO: 47, at least 90% amino acid identitywith amino acids 276-373 of SEQ ID NO: 47 or at least 95% amino acididentity with amino acids 276-373 of SEQ ID NO: 47. In yet other aspectsof this embodiment, a TGFβ4 comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 276-373 of SEQ ID NO: 47,at most 75% amino acid identity with amino acids 276-373 of SEQ ID NO:47, at most 80% amino acid identity with amino acids 276-373 of SEQ IDNO: 47, at most 85% amino acid identity with amino acids 276-373 of SEQID NO: 47, at most 90% amino acid identity with amino acids 276-373 ofSEQ ID NO: 47 or at most 95% amino acid identity with amino acids276-373 of SEQ ID NO: 47.

In other aspects of this embodiment, a TGFβ4 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 276-373 of SEQ ID NO: 47. In other aspects of this embodiment, aTGFβ4 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 276-373 of SEQ ID NO: 47. In yetother aspects of this embodiment, a TGFβ4 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 276-373 of SEQ ID NO: 47. In other aspects of this embodiment, aTGFβ4 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 276-373 of SEQ ID NO: 47. In stillother aspects of this embodiment, a TGFβ4 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 276-373 of SEQ ID NO: 47. In other aspects of this embodiment, aTGFβ4 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 276-373 of SEQ ID NO: 47.

In other aspects of this embodiment, a TGFβ4 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 276-373 of SEQ ID NO: 47. In other aspects of this embodiment, aTGFβ4 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 276-373 of SEQ ID NO: 47. In yetother aspects of this embodiment, a TGFβ4 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids276-373 of SEQ ID NO: 47. In other aspects of this embodiment, a TGFβ4comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 276-373 of SEQ ID NO: 47. In still other aspectsof this embodiment, a TGFβ4 comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 276-373 of SEQID NO: 47. In other aspects of this embodiment, a TGFβ4 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 276-373 of SEQ ID NO: 47.

Another example of an enhanced targeting domain disclosed in the presentspecification is, e.g., a BMPs, such as, e.g., BMP2, BMP3, BMP4, BMP5,BMP6, BMP7, BMP8 or BMP10.

Thus, in an embodiment, an enhanced binding domain derived from a BMP2.In another embodiment, an enhanced binding domain derived from a BMP2 ofSEQ ID NO: 48. In an aspect of this embodiment, an enhanced bindingdomain derived from a BMP2 comprises amino acids 296-396 of SEQ ID NO:48.

In other aspects of this embodiment, a BMP2 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 296-396of SEQ ID NO: 48, at least 75% amino acid identity with amino acids296-396 of SEQ ID NO: 48, at least 80% amino acid identity with aminoacids 296-396 of SEQ ID NO: 48, at least 85% amino acid identity withamino acids 296-396 of SEQ ID NO: 48, at least 90% amino acid identitywith amino acids 296-396 of SEQ ID NO: 48 or at least 95% amino acididentity with amino acids 296-396 of SEQ ID NO: 48. In yet other aspectsof this embodiment, a BMP2 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 296-396 of SEQ ID NO: 48, atmost 75% amino acid identity with amino acids 296-396 of SEQ ID NO: 48,at most 80% amino acid identity with amino acids 296-396 of SEQ ID NO:48, at most 85% amino acid identity with amino acids 296-396 of SEQ IDNO: 48, at most 90% amino acid identity with amino acids 296-396 of SEQID NO: 48 or at most 95% amino acid identity with amino acids 296-396 ofSEQ ID NO: 48.

In other aspects of this embodiment, a BMP2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 296-396 of SEQ ID NO: 48. In other aspects of this embodiment, aBMP2 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 296-396 of SEQ ID NO: 48. In yetother aspects of this embodiment, a BMP2 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids296-396 of SEQ ID NO: 48. In other aspects of this embodiment, a BMP2comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 296-396 of SEQ ID NO: 48. In stillother aspects of this embodiment, a BMP2 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids296-396 of SEQ ID NO: 48. In other aspects of this embodiment, a BMP2comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 296-396 of SEQ ID NO: 48.

In other aspects of this embodiment, a BMP2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 296-396 of SEQ ID NO: 48. In other aspects of this embodiment, aBMP2 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 296-396 of SEQ ID NO: 48. In yetother aspects of this embodiment, a BMP2 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 296-396 ofSEQ ID NO: 48. In other aspects of this embodiment, a BMP2 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 296-396 of SEQ ID NO: 48. In still other aspects of thisembodiment, a BMP2 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 296-396 of SEQ ID NO: 48.In other aspects of this embodiment, a BMP2 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids296-396 of SEQ ID NO: 48.

In another embodiment, an enhanced binding domain derived from a BMP3.In another embodiment, an enhanced binding domain derived from a BMP3 ofSEQ ID NO: 49. In an aspect of this embodiment, an enhanced bindingdomain derived from a BMP3 comprises amino acids 370-472 of SEQ ID NO:49.

In other aspects of this embodiment, a BMP3 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 370-472of SEQ ID NO: 49, at least 75% amino acid identity with amino acids370-472 of SEQ ID NO: 49, at least 80% amino acid identity with aminoacids 370-472 of SEQ ID NO: 49, at least 85% amino acid identity withamino acids 370-472 of SEQ ID NO: 49, at least 90% amino acid identitywith amino acids 370-472 of SEQ ID NO: 49 or at least 95% amino acididentity with amino acids 370-472 of SEQ ID NO: 49. In yet other aspectsof this embodiment, a BMP3 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 370-472 of SEQ ID NO: 49, atmost 75% amino acid identity with amino acids 370-472 of SEQ ID NO: 49,at most 80% amino acid identity with amino acids 370-472 of SEQ ID NO:49, at most 85% amino acid identity with amino acids 370-472 of SEQ IDNO: 49, at most 90% amino acid identity with amino acids 370-472 of SEQID NO: 49 or at most 95% amino acid identity with amino acids 370-472 ofSEQ ID NO: 49.

In other aspects of this embodiment, a BMP3 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 370-472 of SEQ ID NO: 49. In other aspects of this embodiment, aBMP3 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 370-472 of SEQ ID NO: 49. In yetother aspects of this embodiment, a BMP3 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids370-472 of SEQ ID NO: 49. In other aspects of this embodiment, a BMP3comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 370-472 of SEQ ID NO: 49. In stillother aspects of this embodiment, a BMP3 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids370-472 of SEQ ID NO: 49. In other aspects of this embodiment, a BMP3comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 370-472 of SEQ ID NO: 49.

In other aspects of this embodiment, a BMP3 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 370-472 of SEQ ID NO: 49. In other aspects of this embodiment, aBMP3 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 370-472 of SEQ ID NO: 49. In yetother aspects of this embodiment, a BMP3 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 370-472 ofSEQ ID NO: 49. In other aspects of this embodiment, a BMP3 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 370-472 of SEQ ID NO: 49. In still other aspects of thisembodiment, a BMP3 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 370-472 of SEQ ID NO: 49.In other aspects of this embodiment, a BMP3 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids370-472 of SEQ ID NO: 49.

In another embodiment, an enhanced binding domain derived from a BMP4.In another embodiment, an enhanced binding domain derived from a BMP4 ofSEQ ID NO: 50. In an aspect of this embodiment, an enhanced bindingdomain derived from a BMP4 comprises amino acids 309-409 of SEQ ID NO:50.

In other aspects of this embodiment, a BMP4 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 309-409of SEQ ID NO: 50, at least 75% amino acid identity with amino acids309-409 of SEQ ID NO: 50, at least 80% amino acid identity with aminoacids 309-409 of SEQ ID NO: 50, at least 85% amino acid identity withamino acids 309-409 of SEQ ID NO: 50, at least 90% amino acid identitywith amino acids 309-409 of SEQ ID NO: 50 or at least 95% amino acididentity with amino acids 309-409 of SEQ ID NO: 50. In yet other aspectsof this embodiment, a BMP4 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 309-409 of SEQ ID NO: 50, atmost 75% amino acid identity with amino acids 309-409 of SEQ ID NO: 50,at most 80% amino acid identity with amino acids 309-409 of SEQ ID NO:50, at most 85% amino acid identity with amino acids 309-409 of SEQ IDNO: 50, at most 90% amino acid identity with amino acids 309-409 of SEQID NO: 50 or at most 95% amino acid identity with amino acids 309-409 ofSEQ ID NO: 50.

In other aspects of this embodiment, a BMP4 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 309-409 of SEQ ID NO: 50. In other aspects of this embodiment, aBMP4 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 309-409 of SEQ ID NO: 50. In yetother aspects of this embodiment, a BMP4 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids309-409 of SEQ ID NO: 50. In other aspects of this embodiment, a BMP4comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 309-409 of SEQ ID NO: 50. In stillother aspects of this embodiment, a BMP4 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids309-409 of SEQ ID NO: 50. In other aspects of this embodiment, a BMP4comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 309-409 of SEQ ID NO: 50.

In other aspects of this embodiment, a BMP4 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 309-409 of SEQ ID NO: 50. In other aspects of this embodiment, aBMP4 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 309-409 of SEQ ID NO: 50. In yetother aspects of this embodiment, a BMP4 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 309-409 ofSEQ ID NO: 50. In other aspects of this embodiment, a BMP4 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 309-409 of SEQ ID NO: 50. In still other aspects of thisembodiment, a BMP4 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 309-409 of SEQ ID NO: 50.In other aspects of this embodiment, a BMP4 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids309-409 of SEQ ID NO: 50.

In another embodiment, an enhanced binding domain derived from a BMP5.In another embodiment, an enhanced binding domain derived from a BMP5 ofSEQ ID NO: 51. In an aspect of this embodiment, an enhanced bindingdomain derived from a BMP5 comprises amino acids 353-454 or amino acids323-454 of SEQ ID NO: 51.

In other aspects of this embodiment, a BMP5 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 353-454or amino acids 323-454 of SEQ ID NO: 51, at least 75% amino acididentity with amino acids 353-454 or amino acids 323-454 of SEQ ID NO:51, at least 80% amino acid identity with amino acids 353-454 or aminoacids 323-454 of SEQ ID NO: 51, at least 85% amino acid identity withamino acids 353-454 or amino acids 323-454 of SEQ ID NO: 51, at least90% amino acid identity with amino acids 353-454 or amino acids 323-454of SEQ ID NO: 51 or at least 95% amino acid identity with amino acids353-454 or amino acids 323-454 of SEQ ID NO: 51. In yet other aspects ofthis embodiment, a BMP5 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 353-454 or amino acids 323-454of SEQ ID NO: 51, at most 75% amino acid identity with amino acids353-454 or amino acids 323-454 of SEQ ID NO: 51, at most 80% amino acididentity with amino acids 353-454 or amino acids 323-454 of SEQ ID NO:51, at most 85% amino acid identity with amino acids 353-454 or aminoacids 323-454 of SEQ ID NO: 51, at most 90% amino acid identity withamino acids 353-454 or amino acids 323-454 of SEQ ID NO: 51 or at most95% amino acid identity with amino acids 353-454 or amino acids 323-454of SEQ ID NO: 51.

In other aspects of this embodiment, a BMP5 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 353-454 or amino acids 323-454 of SEQ ID NO: 51. In other aspectsof this embodiment, a BMP5 comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 353-454or amino acids 323-454 of SEQ ID NO: 51. In yet other aspects of thisembodiment, a BMP5 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 353-454 or amino acids323-454 of SEQ ID NO: 51. In other aspects of this embodiment, a BMP5comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 353-454 or amino acids 323-454 of SEQID NO: 51. In still other aspects of this embodiment, a BMP5 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 51.In other aspects of this embodiment, a BMP5 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 353-454 or amino acids 323-454 of SEQ ID NO: 51.

In other aspects of this embodiment, a BMP5 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 353-454 or amino acids 323-454 of SEQ ID NO: 51. In other aspectsof this embodiment, a BMP5 comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 353-454 oramino acids 323-454 of SEQ ID NO: 51. In yet other aspects of thisembodiment, a BMP5 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 353-454 or amino acids323-454 of SEQ ID NO: 51. In other aspects of this embodiment, a BMP5comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 51.In still other aspects of this embodiment, a BMP5 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 353-454 or amino acids 323-454 of SEQ ID NO: 51. In otheraspects of this embodiment, a BMP5 comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 353-454 or aminoacids 323-454 of SEQ ID NO: 51.

In another embodiment, an enhanced binding domain derived from a BMP6.In another embodiment, an enhanced binding domain derived from a BMP6 ofSEQ ID NO: 52. In an aspect of this embodiment, an enhanced bindingdomain derived from a BMP6 comprises amino acids 412-513 or amino acids374-513 of SEQ ID NO: 52.

In other aspects of this embodiment, a BMP6 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 412-513or amino acids 374-513 of SEQ ID NO: 52, at least 75% amino acididentity with amino acids 412-513 or amino acids 374-513 of SEQ ID NO:52, at least 80% amino acid identity with amino acids 412-513 or aminoacids 374-513 of SEQ ID NO: 52, at least 85% amino acid identity withamino acids 412-513 or amino acids 374-513 of SEQ ID NO: 52, at least90% amino acid identity with amino acids 412-513 or amino acids 374-513of SEQ ID NO: 52 or at least 95% amino acid identity with amino acids412-513 or amino acids 374-513 of SEQ ID NO: 52. In yet other aspects ofthis embodiment, a BMP6 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 412-513 or amino acids 374-513of SEQ ID NO: 52, at most 75% amino acid identity with amino acids412-513 or amino acids 374-513 of SEQ ID NO: 52, at most 80% amino acididentity with amino acids 412-513 or amino acids 374-513 of SEQ ID NO:52, at most 85% amino acid identity with amino acids 412-513 or aminoacids 374-513 of SEQ ID NO: 52, at most 90% amino acid identity withamino acids 412-513 or amino acids 374-513 of SEQ ID NO: 52 or at most95% amino acid identity with amino acids 412-513 or amino acids 374-513of SEQ ID NO: 52.

In other aspects of this embodiment, a BMP6 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 412-513 or amino acids 374-513 of SEQ ID NO: 52. In other aspectsof this embodiment, a BMP6 comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 412-513or amino acids 374-513 of SEQ ID NO: 52. In yet other aspects of thisembodiment, a BMP6 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 412-513 or amino acids374-513 of SEQ ID NO: 52. In other aspects of this embodiment, a BMP6comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 412-513 or amino acids 374-513 of SEQID NO: 52. In still other aspects of this embodiment, a BMP6 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 52.In other aspects of this embodiment, a BMP6 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 412-513 or amino acids 374-513 of SEQ ID NO: 52.

In other aspects of this embodiment, a BMP6 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 412-513 or amino acids 374-513 of SEQ ID NO: 52. In other aspectsof this embodiment, a BMP6 comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 412-513 oramino acids 374-513 of SEQ ID NO: 52. In yet other aspects of thisembodiment, a BMP6 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 412-513 or amino acids374-513 of SEQ ID NO: 52. In other aspects of this embodiment, a BMP6comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 52.In still other aspects of this embodiment, a BMP6 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 412-513 or amino acids 374-513 of SEQ ID NO: 52. In otheraspects of this embodiment, a BMP6 comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 412-513 or aminoacids 374-513 of SEQ ID NO: 52.

In another embodiment, an enhanced binding domain derived from a BMP7.In another embodiment, an enhanced binding domain derived from a BMP7 ofSEQ ID NO: 53. In an aspect of this embodiment, an enhanced bindingdomain derived from a BMP7 comprises amino acids 330-431 or amino acids293-431 of SEQ ID NO: 53.

In other aspects of this embodiment, a BMP7 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 330-431or amino acids 293-431 of SEQ ID NO: 53, at least 75% amino acididentity with amino acids 330-431 or amino acids 293-431 of SEQ ID NO:53, at least 80% amino acid identity with amino acids 330-431 or aminoacids 293-431 of SEQ ID NO: 53, at least 85% amino acid identity withamino acids 330-431 or amino acids 293-431 of SEQ ID NO: 53, at least90% amino acid identity with amino acids 330-431 or amino acids 293-431of SEQ ID NO: 53 or at least 95% amino acid identity with amino acids330-431 or amino acids 293-431 of SEQ ID NO: 53. In yet other aspects ofthis embodiment, a BMP7 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 330-431 or amino acids 293-431of SEQ ID NO: 53, at most 75% amino acid identity with amino acids330-431 or amino acids 293-431 of SEQ ID NO: 53, at most 80% amino acididentity with amino acids 330-431 or amino acids 293-431 of SEQ ID NO:53, at most 85% amino acid identity with amino acids 330-431 or aminoacids 293-431 of SEQ ID NO: 53, at most 90% amino acid identity withamino acids 330-431 or amino acids 293-431 of SEQ ID NO: 53 or at most95% amino acid identity with amino acids 330-431 or amino acids 293-431of SEQ ID NO: 53.

In other aspects of this embodiment, a BMP7 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 330-431 or amino acids 293-431 of SEQ ID NO: 53. In other aspectsof this embodiment, a BMP7 comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 330-431or amino acids 293-431 of SEQ ID NO: 53. In yet other aspects of thisembodiment, a BMP7 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 330-431 or amino acids293-431 of SEQ ID NO: 53. In other aspects of this embodiment, a BMP7comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 330-431 or amino acids 293-431 of SEQID NO: 53. In still other aspects of this embodiment, a BMP7 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 53.In other aspects of this embodiment, a BMP7 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 330-431 or amino acids 293-431 of SEQ ID NO: 53.

In other aspects of this embodiment, a BMP7 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 330-431 or amino acids 293-431 of SEQ ID NO: 53. In other aspectsof this embodiment, a BMP7 comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 330-431 oramino acids 293-431 of SEQ ID NO: 53. In yet other aspects of thisembodiment, a BMP7 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 330-431 or amino acids293-431 of SEQ ID NO: 53. In other aspects of this embodiment, a BMP7comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 53.In still other aspects of this embodiment, a BMP7 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 330-431 or amino acids 293-431 of SEQ ID NO: 53. In otheraspects of this embodiment, a BMP7 comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 330-431 or aminoacids 293-431 of SEQ ID NO: 53.

In another embodiment, an enhanced binding domain derived from a BMP8.In another embodiment, an enhanced binding domain derived from a BMP8 ofSEQ ID NO: 54. In an aspect of this embodiment, an enhanced bindingdomain derived from a BMP8 comprises amino acids 301-402 of SEQ ID NO:54.

In other aspects of this embodiment, a BMP8 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 301-402of SEQ ID NO: 54, at least 75% amino acid identity with amino acids301-402 of SEQ ID NO: 54, at least 80% amino acid identity with aminoacids 301-402 of SEQ ID NO: 54, at least 85% amino acid identity withamino acids 301-402 of SEQ ID NO: 54, at least 90% amino acid identitywith amino acids 301-402 of SEQ ID NO: 54 or at least 95% amino acididentity with amino acids 301-402 of SEQ ID NO: 54. In yet other aspectsof this embodiment, a BMP8 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 301-402 of SEQ ID NO: 54, atmost 75% amino acid identity with amino acids 301-402 of SEQ ID NO: 54,at most 80% amino acid identity with amino acids 301-402 of SEQ ID NO:54, at most 85% amino acid identity with amino acids 301-402 of SEQ IDNO: 54, at most 90% amino acid identity with amino acids 301-402 of SEQID NO: 54 or at most 95% amino acid identity with amino acids 301-402 ofSEQ ID NO: 54.

In other aspects of this embodiment, a BMP8 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 301-402 of SEQ ID NO: 54. In other aspects of this embodiment, aBMP8 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 301-402 of SEQ ID NO: 54. In yetother aspects of this embodiment, a BMP8 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids301-402 of SEQ ID NO: 54. In other aspects of this embodiment, a BMP8comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 301-402 of SEQ ID NO: 54. In stillother aspects of this embodiment, a BMP8 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids301-402 of SEQ ID NO: 54. In other aspects of this embodiment, a BMP8comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 301-402 of SEQ ID NO: 54.

In other aspects of this embodiment, a BMP8 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 301-402 of SEQ ID NO: 54. In other aspects of this embodiment, aBMP8 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 301-402 of SEQ ID NO: 54. In yetother aspects of this embodiment, a BMP8 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 301-402 ofSEQ ID NO: 54. In other aspects of this embodiment, a BMP8 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 301-402 of SEQ ID NO: 54. In still other aspects of thisembodiment, a BMP8 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 301-402 of SEQ ID NO: 54.In other aspects of this embodiment, a BMP8 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids301-402 of SEQ ID NO: 54.

In another embodiment, an enhanced binding domain derived from a BMP10.In another embodiment, an enhanced binding domain derived from a BMP10of SEQ ID NO: 55. In an aspect of this embodiment, an enhanced bindingdomain derived from a BMP10 comprises amino acids 323-424 of SEQ ID NO:55.

In other aspects of this embodiment, a BMP10 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 323-424of SEQ ID NO: 55, at least 75% amino acid identity with amino acids323-424 of SEQ ID NO: 55, at least 80% amino acid identity with aminoacids 323-424 of SEQ ID NO: 55, at least 85% amino acid identity withamino acids 323-424 of SEQ ID NO: 55, at least 90% amino acid identitywith amino acids 323-424 of SEQ ID NO: 55 or at least 95% amino acididentity with amino acids 323-424 of SEQ ID NO: 55. In yet other aspectsof this embodiment, a BMP10 comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 323-424 of SEQ ID NO: 55,at most 75% amino acid identity with amino acids 323-424 of SEQ ID NO:55, at most 80% amino acid identity with amino acids 323-424 of SEQ IDNO: 55, at most 85% amino acid identity with amino acids 323-424 of SEQID NO: 55, at most 90% amino acid identity with amino acids 323-424 ofSEQ ID NO: 55 or at most 95% amino acid identity with amino acids323-424 of SEQ ID NO: 55.

In other aspects of this embodiment, a BMP10 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 323-424 of SEQ ID NO: 55. In other aspects of this embodiment, aBMP10 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 323-424 of SEQ ID NO: 55. In yetother aspects of this embodiment, a BMP10 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 323-424 of SEQ ID NO: 55. In other aspects of this embodiment, aBMP10 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 323-424 of SEQ ID NO: 55. In stillother aspects of this embodiment, a BMP10 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 323-424 of SEQ ID NO: 55. In other aspects of this embodiment, aBMP10 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 323-424 of SEQ ID NO: 55.

In other aspects of this embodiment, a BMP10 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 323-424 of SEQ ID NO: 55. In other aspects of this embodiment, aBMP10 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 323-424 of SEQ ID NO: 55. In yetother aspects of this embodiment, a BMP10 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids323-424 of SEQ ID NO: 55. In other aspects of this embodiment, a BMP10comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 323-424 of SEQ ID NO: 55. In still other aspectsof this embodiment, a BMP10 comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 323-424 of SEQID NO: 55. In other aspects of this embodiment, a BMP10 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 323-424 of SEQ ID NO: 55.

Another example of an enhanced targeting domain disclosed in the presentspecification is, e.g., a GFPs, such as, e.g., GDF1, GDF2, GDF3, GDF5,GDF6, GDF7, GDF8, GDF10, GDF11 or GDF15.

Thus, in an embodiment, an enhanced binding domain derived from a GDF1.In another embodiment, an enhanced binding domain derived from a GDF1 ofSEQ ID NO: 56. In an aspect of this embodiment, an enhanced bindingdomain derived from a GDF1 comprises amino acids 267-372 of SEQ ID NO:56.

In other aspects of this embodiment, a GDF1 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 267-372of SEQ ID NO: 56, at least 75% amino acid identity with amino acids267-372 of SEQ ID NO: 56, at least 80% amino acid identity with aminoacids 267-372 of SEQ ID NO: 56, at least 85% amino acid identity withamino acids 267-372 of SEQ ID NO: 56, at least 90% amino acid identitywith amino acids 267-372 of SEQ ID NO: 56 or at least 95% amino acididentity with amino acids 267-372 of SEQ ID NO: 56. In yet other aspectsof this embodiment, a GDF1 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 267-372 of SEQ ID NO: 56, atmost 75% amino acid identity with amino acids 267-372 of SEQ ID NO: 56,at most 80% amino acid identity with amino acids 267-372 of SEQ ID NO:56, at most 85% amino acid identity with amino acids 267-372 of SEQ IDNO: 56, at most 90% amino acid identity with amino acids 267-372 of SEQID NO: 56 or at most 95% amino acid identity with amino acids 267-372 ofSEQ ID NO: 56.

In other aspects of this embodiment, a GDF1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 267-372 of SEQ ID NO: 56. In other aspects of this embodiment, aGDF1 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 267-372 of SEQ ID NO: 56. In yetother aspects of this embodiment, a GDF1 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids267-372 of SEQ ID NO: 56. In other aspects of this embodiment, a GDF1comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 267-372 of SEQ ID NO: 56. In stillother aspects of this embodiment, a GDF1 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids267-372 of SEQ ID NO: 56. In other aspects of this embodiment, a GDF1comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 267-372 of SEQ ID NO: 56.

In other aspects of this embodiment, a GDF1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 267-372 of SEQ ID NO: 56. In other aspects of this embodiment, aGDF1 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 267-372 of SEQ ID NO: 56. In yetother aspects of this embodiment, a GDF1 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 267-372 ofSEQ ID NO: 56. In other aspects of this embodiment, a GDF1 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 267-372 of SEQ ID NO: 56. In still other aspects of thisembodiment, a GDF1 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 267-372 of SEQ ID NO: 56.In other aspects of this embodiment, a GDF1 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids267-372 of SEQ ID NO: 56.

In another embodiment, an enhanced binding domain derived from a GDF2.In another embodiment, an enhanced binding domain derived from a GDF2 ofSEQ ID NO: 57. In an aspect of this embodiment, an enhanced bindingdomain derived from a GDF2 comprises amino acids 327-429 of SEQ ID NO:57.

In other aspects of this embodiment, a GDF2 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 327-429of SEQ ID NO: 57, at least 75% amino acid identity with amino acids327-429 of SEQ ID NO: 57, at least 80% amino acid identity with aminoacids 327-429 of SEQ ID NO: 57, at least 85% amino acid identity withamino acids 327-429 of SEQ ID NO: 57, at least 90% amino acid identitywith amino acids 327-429 of SEQ ID NO: 57 or at least 95% amino acididentity with amino acids 327-429 of SEQ ID NO: 57. In yet other aspectsof this embodiment, a GDF2 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 327-429 of SEQ ID NO: 57, atmost 75% amino acid identity with amino acids 327-429 of SEQ ID NO: 57,at most 80% amino acid identity with amino acids 327-429 of SEQ ID NO:57, at most 85% amino acid identity with amino acids 327-429 of SEQ IDNO: 57, at most 90% amino acid identity with amino acids 327-429 of SEQID NO: 57 or at most 95% amino acid identity with amino acids 327-429 ofSEQ ID NO: 57.

In other aspects of this embodiment, a GDF2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 327-429 of SEQ ID NO: 57. In other aspects of this embodiment, aGDF2 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 327-429 of SEQ ID NO: 57. In yetother aspects of this embodiment, a GDF2 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids327-429 of SEQ ID NO: 57. In other aspects of this embodiment, a GDF2comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 327-429 of SEQ ID NO: 57. In stillother aspects of this embodiment, a GDF2 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids327-429 of SEQ ID NO: 57. In other aspects of this embodiment, a GDF2comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 327-429 of SEQ ID NO: 57.

In other aspects of this embodiment, a GDF2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 327-429 of SEQ ID NO: 57. In other aspects of this embodiment, aGDF2 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 327-429 of SEQ ID NO: 57. In yetother aspects of this embodiment, a GDF2 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 327-429 ofSEQ ID NO: 57. In other aspects of this embodiment, a GDF2 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 327-429 of SEQ ID NO: 57. In still other aspects of thisembodiment, a GDF2 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 327-429 of SEQ ID NO: 57.In other aspects of this embodiment, a GDF2 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids327-429 of SEQ ID NO: 57.

In another embodiment, an enhanced binding domain derived from a GDF3.In another embodiment, an enhanced binding domain derived from a GDF3 ofSEQ ID NO: 58. In an aspect of this embodiment, an enhanced bindingdomain derived from a GDF3 comprises amino acids 264-364 of SEQ ID NO:58.

In other aspects of this embodiment, a GDF3 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 264-364of SEQ ID NO: 58, at least 75% amino acid identity with amino acids264-364 of SEQ ID NO: 58, at least 80% amino acid identity with aminoacids 264-364 of SEQ ID NO: 58, at least 85% amino acid identity withamino acids 264-364 of SEQ ID NO: 58, at least 90% amino acid identitywith amino acids 264-364 of SEQ ID NO: 58 or at least 95% amino acididentity with amino acids 264-364 of SEQ ID NO: 58. In yet other aspectsof this embodiment, a GDF3 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 264-364 of SEQ ID NO: 58, atmost 75% amino acid identity with amino acids 264-364 of SEQ ID NO: 58,at most 80% amino acid identity with amino acids 264-364 of SEQ ID NO:58, at most 85% amino acid identity with amino acids 264-364 of SEQ IDNO: 58, at most 90% amino acid identity with amino acids 264-364 of SEQID NO: 58 or at most 95% amino acid identity with amino acids 264-364 ofSEQ ID NO: 58.

In other aspects of this embodiment, a GDF3 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 264-364 of SEQ ID NO: 58. In other aspects of this embodiment, aGDF3 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 264-364 of SEQ ID NO: 58. In yetother aspects of this embodiment, a GDF3 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids264-364 of SEQ ID NO: 58. In other aspects of this embodiment, a GDF3comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 264-364 of SEQ ID NO: 58. In stillother aspects of this embodiment, a GDF3 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids264-364 of SEQ ID NO: 58. In other aspects of this embodiment, a GDF3comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 264-364 of SEQ ID NO: 58.

In other aspects of this embodiment, a GDF3 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 264-364 of SEQ ID NO: 58. In other aspects of this embodiment, aGDF3 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 264-364 of SEQ ID NO: 58. In yetother aspects of this embodiment, a GDF3 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 264-364 ofSEQ ID NO: 58. In other aspects of this embodiment, a GDF3 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 264-364 of SEQ ID NO: 58. In still other aspects of thisembodiment, a GDF3 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 264-364 of SEQ ID NO: 58.In other aspects of this embodiment, a GDF3 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids264-364 of SEQ ID NO: 58.

In another embodiment, an enhanced binding domain derived from a GDF5.In another embodiment, an enhanced binding domain derived from a GDF5 ofSEQ ID NO: 59. In an aspect of this embodiment, an enhanced bindingdomain derived from a GDF5 comprises amino acids 400-501 of SEQ ID NO:59.

In other aspects of this embodiment, a GDF5 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 400-501of SEQ ID NO: 59, at least 75% amino acid identity with amino acids400-501 of SEQ ID NO: 59, at least 80% amino acid identity with aminoacids 400-501 of SEQ ID NO: 59, at least 85% amino acid identity withamino acids 400-501 of SEQ ID NO: 59, at least 90% amino acid identitywith amino acids 400-501 of SEQ ID NO: 59 or at least 95% amino acididentity with amino acids 400-501 of SEQ ID NO: 59. In yet other aspectsof this embodiment, a GDF5 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 400-501 of SEQ ID NO: 59, atmost 75% amino acid identity with amino acids 400-501 of SEQ ID NO: 59,at most 80% amino acid identity with amino acids 400-501 of SEQ ID NO:59, at most 85% amino acid identity with amino acids 400-501 of SEQ IDNO: 59, at most 90% amino acid identity with amino acids 400-501 of SEQID NO: 59 or at most 95% amino acid identity with amino acids 400-501 ofSEQ ID NO: 59.

In other aspects of this embodiment, a GDF5 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 400-501 of SEQ ID NO: 59. In other aspects of this embodiment, aGDF5 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 400-501 of SEQ ID NO: 59. In yetother aspects of this embodiment, a GDF5 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids400-501 of SEQ ID NO: 59. In other aspects of this embodiment, a GDF5comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 400-501 of SEQ ID NO: 59. In stillother aspects of this embodiment, a GDF5 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids400-501 of SEQ ID NO: 59. In other aspects of this embodiment, a GDF5comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 400-501 of SEQ ID NO: 59.

In other aspects of this embodiment, a GDF5 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 400-501 of SEQ ID NO: 59. In other aspects of this embodiment, aGDF5 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 400-501 of SEQ ID NO: 59. In yetother aspects of this embodiment, a GDF5 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 400-501 ofSEQ ID NO: 59. In other aspects of this embodiment, a GDF5 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 400-501 of SEQ ID NO: 59. In still other aspects of thisembodiment, a GDF5 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 400-501 of SEQ ID NO: 59.In other aspects of this embodiment, a GDF5 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids400-501 of SEQ ID NO: 59.

In another embodiment, an enhanced binding domain derived from a GDF6.In another embodiment, an enhanced binding domain derived from a GDF6 ofSEQ ID NO: 60. In an aspect of this embodiment, an enhanced bindingdomain derived from a GDF6 comprises amino acids 354-455 of SEQ ID NO:60.

In other aspects of this embodiment, a GDF6 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 354-455of SEQ ID NO: 60, at least 75% amino acid identity with amino acids354-455 of SEQ ID NO: 60, at least 80% amino acid identity with aminoacids 354-455 of SEQ ID NO: 60, at least 85% amino acid identity withamino acids 354-455 of SEQ ID NO: 60, at least 90% amino acid identitywith amino acids 354-455 of SEQ ID NO: 60 or at least 95% amino acididentity with amino acids 354-455 of SEQ ID NO: 60. In yet other aspectsof this embodiment, a GDF6 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 354-455 of SEQ ID NO: 60, atmost 75% amino acid identity with amino acids 354-455 of SEQ ID NO: 60,at most 80% amino acid identity with amino acids 354-455 of SEQ ID NO:60, at most 85% amino acid identity with amino acids 354-455 of SEQ IDNO: 60, at most 90% amino acid identity with amino acids 354-455 of SEQID NO: 60 or at most 95% amino acid identity with amino acids 354-455 ofSEQ ID NO: 60.

In other aspects of this embodiment, a GDF6 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 354-455 of SEQ ID NO: 60. In other aspects of this embodiment, aGDF6 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 354-455 of SEQ ID NO: 60. In yetother aspects of this embodiment, a GDF6 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids354-455 of SEQ ID NO: 60. In other aspects of this embodiment, a GDF6comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 354-455 of SEQ ID NO: 60. In stillother aspects of this embodiment, a GDF6 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids354-455 of SEQ ID NO: 60. In other aspects of this embodiment, a GDF6comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 354-455 of SEQ ID NO: 60.

In other aspects of this embodiment, a GDF6 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 354-455 of SEQ ID NO: 60. In other aspects of this embodiment, aGDF6 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 354-455 of SEQ ID NO: 60. In yetother aspects of this embodiment, a GDF6 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 354-455 ofSEQ ID NO: 60. In other aspects of this embodiment, a GDF6 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 354-455 of SEQ ID NO: 60. In still other aspects of thisembodiment, a GDF6 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 354-455 of SEQ ID NO: 60.In other aspects of this embodiment, a GDF6 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids354-455 of SEQ ID NO: 60.

In another embodiment, an enhanced binding domain derived from a GDF7.In another embodiment, an enhanced binding domain derived from a GDF7 ofSEQ ID NO: 61. In an aspect of this embodiment, an enhanced bindingdomain derived from a GDF7 comprises amino acids 352-450 of SEQ ID NO:61.

In other aspects of this embodiment, a GDF7 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 352-450of SEQ ID NO: 61, at least 75% amino acid identity with amino acids352-450 of SEQ ID NO: 61, at least 80% amino acid identity with aminoacids 352-450 of SEQ ID NO: 61, at least 85% amino acid identity withamino acids 352-450 of SEQ ID NO: 61, at least 90% amino acid identitywith amino acids 352-450 of SEQ ID NO: 61 or at least 95% amino acididentity with amino acids 352-450 of SEQ ID NO: 61. In yet other aspectsof this embodiment, a GDF7 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 352-450 of SEQ ID NO: 61, atmost 75% amino acid identity with amino acids 352-450 of SEQ ID NO: 61,at most 80% amino acid identity with amino acids 352-450 of SEQ ID NO:61, at most 85% amino acid identity with amino acids 352-450 of SEQ IDNO: 61, at most 90% amino acid identity with amino acids 352-450 of SEQID NO: 61 or at most 95% amino acid identity with amino acids 352-450 ofSEQ ID NO: 61.

In other aspects of this embodiment, a GDF7 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 352-450 of SEQ ID NO: 61. In other aspects of this embodiment, aGDF7 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 352-450 of SEQ ID NO: 61. In yetother aspects of this embodiment, a GDF7 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids352-450 of SEQ ID NO: 61. In other aspects of this embodiment, a GDF7comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 352-450 of SEQ ID NO: 61. In stillother aspects of this embodiment, a GDF7 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids352-450 of SEQ ID NO: 61. In other aspects of this embodiment, a GDF7comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 352-450 of SEQ ID NO: 61.

In other aspects of this embodiment, a GDF7 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 352-450 of SEQ ID NO: 61. In other aspects of this embodiment, aGDF7 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 352-450 of SEQ ID NO: 61. In yetother aspects of this embodiment, a GDF7 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 352-450 ofSEQ ID NO: 61. In other aspects of this embodiment, a GDF7 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 352-450 of SEQ ID NO: 61. In still other aspects of thisembodiment, a GDF7 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 352-450 of SEQ ID NO: 61.In other aspects of this embodiment, a GDF7 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids352-450 of SEQ ID NO: 61.

In another embodiment, an enhanced binding domain derived from a GDF8.In another embodiment, an enhanced binding domain derived from a GDF8 ofSEQ ID NO: 62. In an aspect of this embodiment, an enhanced bindingdomain derived from a GDF8 comprises amino acids 281-375 of SEQ ID NO:62.

In other aspects of this embodiment, a GDF8 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 281-375of SEQ ID NO: 62, at least 75% amino acid identity with amino acids281-375 of SEQ ID NO: 62, at least 80% amino acid identity with aminoacids 281-375 of SEQ ID NO: 62, at least 85% amino acid identity withamino acids 281-375 of SEQ ID NO: 62, at least 90% amino acid identitywith amino acids 281-375 of SEQ ID NO: 62 or at least 95% amino acididentity with amino acids 281-375 of SEQ ID NO: 62. In yet other aspectsof this embodiment, a GDF8 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 281-375 of SEQ ID NO: 62, atmost 75% amino acid identity with amino acids 281-375 of SEQ ID NO: 62,at most 80% amino acid identity with amino acids 281-375 of SEQ ID NO:62, at most 85% amino acid identity with amino acids 281-375 of SEQ IDNO: 62, at most 90% amino acid identity with amino acids 281-375 of SEQID NO: 62 or at most 95% amino acid identity with amino acids 281-375 ofSEQ ID NO: 62.

In other aspects of this embodiment, a GDF8 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 281-375 of SEQ ID NO: 62. In other aspects of this embodiment, aGDF8 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 281-375 of SEQ ID NO: 62. In yetother aspects of this embodiment, a GDF8 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids281-375 of SEQ ID NO: 62. In other aspects of this embodiment, a GDF8comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 281-375 of SEQ ID NO: 62. In stillother aspects of this embodiment, a GDF8 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids281-375 of SEQ ID NO: 62. In other aspects of this embodiment, a GDF8comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 281-375 of SEQ ID NO: 62.

In other aspects of this embodiment, a GDF8 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 281-375 of SEQ ID NO: 62. In other aspects of this embodiment, aGDF8 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 281-375 of SEQ ID NO: 62. In yetother aspects of this embodiment, a GDF8 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 281-375 ofSEQ ID NO: 62. In other aspects of this embodiment, a GDF8 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 281-375 of SEQ ID NO: 62. In still other aspects of thisembodiment, a GDF8 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 281-375 of SEQ ID NO: 62.In other aspects of this embodiment, a GDF8 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids281-375 of SEQ ID NO: 62.

In another embodiment, an enhanced binding domain derived from a GDF10.In another embodiment, an enhanced binding domain derived from a GDF10of SEQ ID NO: 63. In an aspect of this embodiment, an enhanced bindingdomain derived from a GDF10 comprises amino acids 376-478 of SEQ ID NO:63.

In other aspects of this embodiment, a GDF10 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 376-478of SEQ ID NO: 63, at least 75% amino acid identity with amino acids376-478 of SEQ ID NO: 63, at least 80% amino acid identity with aminoacids 376-478 of SEQ ID NO: 63, at least 85% amino acid identity withamino acids 376-478 of SEQ ID NO: 63, at least 90% amino acid identitywith amino acids 376-478 of SEQ ID NO: 63 or at least 95% amino acididentity with amino acids 376-478 of SEQ ID NO: 63. In yet other aspectsof this embodiment, a GDF10 comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 376-478 of SEQ ID NO: 63,at most 75% amino acid identity with amino acids 376-478 of SEQ ID NO:63, at most 80% amino acid identity with amino acids 376-478 of SEQ IDNO: 63, at most 85% amino acid identity with amino acids 376-478 of SEQID NO: 63, at most 90% amino acid identity with amino acids 376-478 ofSEQ ID NO: 63 or at most 95% amino acid identity with amino acids376-478 of SEQ ID NO: 63.

In other aspects of this embodiment, a GDF10 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 376-478 of SEQ ID NO: 63. In other aspects of this embodiment, aGDF10 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 376-478 of SEQ ID NO: 63. In yetother aspects of this embodiment, a GDF10 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 376-478 of SEQ ID NO: 63. In other aspects of this embodiment, aGDF10 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 376-478 of SEQ ID NO: 63. In stillother aspects of this embodiment, a GDF10 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 376-478 of SEQ ID NO: 63. In other aspects of this embodiment, aGDF10 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 376-478 of SEQ ID NO: 63.

In other aspects of this embodiment, a GDF10 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 376-478 of SEQ ID NO: 63. In other aspects of this embodiment, aGDF10 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 376-478 of SEQ ID NO: 63. In yetother aspects of this embodiment, a GDF10 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids376-478 of SEQ ID NO: 63. In other aspects of this embodiment, a GDF10comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 376-478 of SEQ ID NO: 63. In still other aspectsof this embodiment, a GDF10 comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 376-478 of SEQID NO: 63. In other aspects of this embodiment, a GDF10 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 376-478 of SEQ ID NO: 63.

In another embodiment, an enhanced binding domain derived from a GDF11.In another embodiment, an enhanced binding domain derived from a GDF11of SEQ ID NO: 64. In an aspect of this embodiment, an enhanced bindingdomain derived from a GDF11 comprises amino acids 313-407 of SEQ ID NO:64.

In other aspects of this embodiment, a GDF11 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 313-407of SEQ ID NO: 64, at least 75% amino acid identity with amino acids313-407 of SEQ ID NO: 64, at least 80% amino acid identity with aminoacids 313-407 of SEQ ID NO: 64, at least 85% amino acid identity withamino acids 313-407 of SEQ ID NO: 64, at least 90% amino acid identitywith amino acids 313-407 of SEQ ID NO: 64 or at least 95% amino acididentity with amino acids 313-407 of SEQ ID NO: 64. In yet other aspectsof this embodiment, a GDF11 comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 313-407 of SEQ ID NO: 64,at most 75% amino acid identity with amino acids 313-407 of SEQ ID NO:64, at most 80% amino acid identity with amino acids 313-407 of SEQ IDNO: 64, at most 85% amino acid identity with amino acids 313-407 of SEQID NO: 64, at most 90% amino acid identity with amino acids 313-407 ofSEQ ID NO: 64 or at most 95% amino acid identity with amino acids313-407 of SEQ ID NO: 64.

In other aspects of this embodiment, a GDF11 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 313-407 of SEQ ID NO: 64. In other aspects of this embodiment, aGDF11 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 313-407 of SEQ ID NO: 64. In yetother aspects of this embodiment, a GDF11 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 313-407 of SEQ ID NO: 64. In other aspects of this embodiment, aGDF11 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 313-407 of SEQ ID NO: 64. In stillother aspects of this embodiment, a GDF11 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 313-407 of SEQ ID NO: 64. In other aspects of this embodiment, aGDF11 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 313-407 of SEQ ID NO: 64.

In other aspects of this embodiment, a GDF11 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 313-407 of SEQ ID NO: 64. In other aspects of this embodiment, aGDF11 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 313-407 of SEQ ID NO: 64. In yetother aspects of this embodiment, a GDF11 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids313-407 of SEQ ID NO: 64. In other aspects of this embodiment, a GDF11comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 313-407 of SEQ ID NO: 64. In still other aspectsof this embodiment, a GDF11 comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 313-407 of SEQID NO: 64. In other aspects of this embodiment, a GDF11 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 313-407 of SEQ ID NO: 64.

In another embodiment, an enhanced binding domain derived from a GDF15.In another embodiment, an enhanced binding domain derived from a GDF15of SEQ ID NO: 65. In an aspect of this embodiment, an enhanced bindingdomain derived from a GDF15 comprises amino acids 211-308 of SEQ ID NO:65.

In other aspects of this embodiment, a GDF15 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 211-308of SEQ ID NO: 65, at least 75% amino acid identity with amino acids211-308 of SEQ ID NO: 65, at least 80% amino acid identity with aminoacids 211-308 of SEQ ID NO: 65, at least 85% amino acid identity withamino acids 211-308 of SEQ ID NO: 65, at least 90% amino acid identitywith amino acids 211-308 of SEQ ID NO: 65 or at least 95% amino acididentity with amino acids 211-308 of SEQ ID NO: 65. In yet other aspectsof this embodiment, a GDF15 comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 211-308 of SEQ ID NO: 65,at most 75% amino acid identity with amino acids 211-308 of SEQ ID NO:65, at most 80% amino acid identity with amino acids 211-308 of SEQ IDNO: 65, at most 85% amino acid identity with amino acids 211-308 of SEQID NO: 65, at most 90% amino acid identity with amino acids 211-308 ofSEQ ID NO: 65 or at most 95% amino acid identity with amino acids211-308 of SEQ ID NO: 65.

In other aspects of this embodiment, a GDF15 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 211-308 of SEQ ID NO: 65. In other aspects of this embodiment, aGDF15 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 211-308 of SEQ ID NO: 65. In yetother aspects of this embodiment, a GDF15 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 211-308 of SEQ ID NO: 65. In other aspects of this embodiment, aGDF15 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 211-308 of SEQ ID NO: 65. In stillother aspects of this embodiment, a GDF15 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 211-308 of SEQ ID NO: 65. In other aspects of this embodiment, aGDF15 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 211-308 of SEQ ID NO: 65.

In other aspects of this embodiment, a GDF15 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 211-308 of SEQ ID NO: 65. In other aspects of this embodiment, aGDF15 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 211-308 of SEQ ID NO: 65. In yetother aspects of this embodiment, a GDF15 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids211-308 of SEQ ID NO: 65. In other aspects of this embodiment, a GDF15comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 211-308 of SEQ ID NO: 65. In still other aspectsof this embodiment, a GDF15 comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 211-308 of SEQID NO: 65. In other aspects of this embodiment, a GDF15 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 211-308 of SEQ ID NO: 65.

Another example of an enhanced targeting domain disclosed in the presentspecification is, e.g., an activin A, an activin B, an activin C, anactivin E or an inhibin A.

In another embodiment, an enhanced binding domain derived from anActivin A. In another embodiment, an enhanced binding domain derivedfrom an Activin A of SEQ ID NO: 66. In an aspect of this embodiment, anenhanced binding domain derived from an Activin A comprises amino acids321-426 of SEQ ID NO: 66.

In other aspects of this embodiment, an Activin A comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 321-426 of SEQ ID NO: 66, at least 75% amino acid identity withamino acids 321-426 of SEQ ID NO: 66, at least 80% amino acid identitywith amino acids 321-426 of SEQ ID NO: 66, at least 85% amino acididentity with amino acids 321-426 of SEQ ID NO: 66, at least 90% aminoacid identity with amino acids 321-426 of SEQ ID NO: 66 or at least 95%amino acid identity with amino acids 321-426 of SEQ ID NO: 66. In yetother aspects of this embodiment, an Activin A comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 321-426of SEQ ID NO: 66, at most 75% amino acid identity with amino acids321-426 of SEQ ID NO: 66, at most 80% amino acid identity with aminoacids 321-426 of SEQ ID NO: 66, at most 85% amino acid identity withamino acids 321-426 of SEQ ID NO: 66, at most 90% amino acid identitywith amino acids 321-426 of SEQ ID NO: 66 or at most 95% amino acididentity with amino acids 321-426 of SEQ ID NO: 66.

In other aspects of this embodiment, an Activin A comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid substitutionsrelative to amino acids 321-426 of SEQ ID NO: 66. In other aspects ofthis embodiment, an Activin A comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 321-426of SEQ ID NO: 66. In yet other aspects of this embodiment, an Activin Acomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 321-426 of SEQ ID NO: 66. In otheraspects of this embodiment, an Activin A comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids321-426 of SEQ ID NO: 66. In still other aspects of this embodiment, anActivin A comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 321-426 of SEQ ID NO: 66. In otheraspects of this embodiment, an Activin A comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids321-426 of SEQ ID NO: 66.

In other aspects of this embodiment, an Activin A comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to amino acids 321-426 of SEQ ID NO: 66. In other aspects ofthis embodiment, an Activin A comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 321-426 ofSEQ ID NO: 66. In yet other aspects of this embodiment, an Activin Acomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 321-426 of SEQ ID NO: 66. In other aspects ofthis embodiment, an Activin A comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 321-426 of SEQID NO: 66. In still other aspects of this embodiment, an Activin Acomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 321-426 of SEQ ID NO: 66. In other aspects ofthis embodiment, an Activin A comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 321-426 of SEQID NO: 66.

In another embodiment, an enhanced binding domain derived from anActivin B. In another embodiment, an enhanced binding domain derivedfrom an Activin B of SEQ ID NO: 67. In an aspect of this embodiment, anenhanced binding domain derived from an Activin B comprises amino acids303-406 of SEQ ID NO: 67.

In other aspects of this embodiment, an Activin B comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 303-406 of SEQ ID NO: 67, at least 75% amino acid identity withamino acids 303-406 of SEQ ID NO: 67, at least 80% amino acid identitywith amino acids 303-406 of SEQ ID NO: 67, at least 85% amino acididentity with amino acids 303-406 of SEQ ID NO: 67, at least 90% aminoacid identity with amino acids 303-406 of SEQ ID NO: 67 or at least 95%amino acid identity with amino acids 303-406 of SEQ ID NO: 67. In yetother aspects of this embodiment, an Activin B comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 303-406of SEQ ID NO: 67, at most 75% amino acid identity with amino acids303-406 of SEQ ID NO: 67, at most 80% amino acid identity with aminoacids 303-406 of SEQ ID NO: 67, at most 85% amino acid identity withamino acids 303-406 of SEQ ID NO: 67, at most 90% amino acid identitywith amino acids 303-406 of SEQ ID NO: 67 or at most 95% amino acididentity with amino acids 303-406 of SEQ ID NO: 67.

In other aspects of this embodiment, an Activin B comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid substitutionsrelative to amino acids 303-406 of SEQ ID NO: 67. In other aspects ofthis embodiment, an Activin B comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 303-406of SEQ ID NO: 67. In yet other aspects of this embodiment, an Activin Bcomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 303-406 of SEQ ID NO: 67. In otheraspects of this embodiment, an Activin B comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids303-406 of SEQ ID NO: 67. In still other aspects of this embodiment, anActivin B comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 303-406 of SEQ ID NO: 67. In otheraspects of this embodiment, an Activin B comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids303-406 of SEQ ID NO: 67.

In other aspects of this embodiment, an Activin B comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to amino acids 303-406 of SEQ ID NO: 67. In other aspects ofthis embodiment, an Activin B comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 303-406 ofSEQ ID NO: 67. In yet other aspects of this embodiment, an Activin Bcomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 303-406 of SEQ ID NO: 67. In other aspects ofthis embodiment, an Activin B comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 303-406 of SEQID NO: 67. In still other aspects of this embodiment, an Activin Bcomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 303-406 of SEQ ID NO: 67. In other aspects ofthis embodiment, an Activin B comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 303-406 of SEQID NO: 67.

In another embodiment, an enhanced binding domain derived from anActivin C. In another embodiment, an enhanced binding domain derivedfrom an Activin C of SEQ ID NO: 68. In an aspect of this embodiment, anenhanced binding domain derived from an Activin C comprises amino acids247-352 or amino acids 237-352 of SEQ ID NO: 68.

In other aspects of this embodiment, an Activin C comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 247-352 or amino acids 237-352 of SEQ ID NO: 68, at least 75%amino acid identity with amino acids 247-352 or amino acids 237-352 ofSEQ ID NO: 68, at least 80% amino acid identity with amino acids 247-352or amino acids 237-352 of SEQ ID NO: 68, at least 85% amino acididentity with amino acids 247-352 or amino acids 237-352 of SEQ ID NO:68, at least 90% amino acid identity with amino acids 247-352 or aminoacids 237-352 of SEQ ID NO: 68 or at least 95% amino acid identity withamino acids 247-352 or amino acids 237-352 of SEQ ID NO: 68. In yetother aspects of this embodiment, an Activin C comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 247-352or amino acids 237-352 of SEQ ID NO: 68, at most 75% amino acid identitywith amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 68, atmost 80% amino acid identity with amino acids 247-352 or amino acids237-352 of SEQ ID NO: 68, at most 85% amino acid identity with aminoacids 247-352 or amino acids 237-352 of SEQ ID NO: 68, at most 90% aminoacid identity with amino acids 247-352 or amino acids 237-352 of SEQ IDNO: 68 or at most 95% amino acid identity with amino acids 247-352 oramino acids 237-352 of SEQ ID NO: 68.

In other aspects of this embodiment, an Activin C comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid substitutionsrelative to amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 68.In other aspects of this embodiment, an Activin C comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid substitutionsrelative to amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 68.In yet other aspects of this embodiment, an Activin C comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 68.In other aspects of this embodiment, an Activin C comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 68.In still other aspects of this embodiment, an Activin C comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 68.In other aspects of this embodiment, an Activin C comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 68.

In other aspects of this embodiment, an Activin C comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 68.In other aspects of this embodiment, an Activin C comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to amino acids 247-352 or amino acids 237-352 of SEQ ID NO: 68.In yet other aspects of this embodiment, an Activin C comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 247-352 or amino acids 237-352 of SEQ ID NO: 68. In otheraspects of this embodiment, an Activin C comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 247-352 oramino acids 237-352 of SEQ ID NO: 68. In still other aspects of thisembodiment, an Activin C comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 247-352 or aminoacids 237-352 of SEQ ID NO: 68. In other aspects of this embodiment, anActivin C comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid additions relative to amino acids 247-352 or amino acids 237-352 ofSEQ ID NO: 68.

In another embodiment, an enhanced binding domain derived from anActivin E. In another embodiment, an enhanced binding domain derivedfrom an Activin E of SEQ ID NO: 69. In an aspect of this embodiment, anenhanced binding domain derived from an Activin E comprises amino acids247-350 of SEQ ID NO: 69.

In other aspects of this embodiment, an Activin E comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 247-350 of SEQ ID NO: 69, at least 75% amino acid identity withamino acids 247-350 of SEQ ID NO: 69, at least 80% amino acid identitywith amino acids 247-350 of SEQ ID NO: 69, at least 85% amino acididentity with amino acids 247-350 of SEQ ID NO: 69, at least 90% aminoacid identity with amino acids 247-350 of SEQ ID NO: 69 or at least 95%amino acid identity with amino acids 247-350 of SEQ ID NO: 69. In yetother aspects of this embodiment, an Activin E comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 247-350of SEQ ID NO: 69, at most 75% amino acid identity with amino acids247-350 of SEQ ID NO: 69, at most 80% amino acid identity with aminoacids 247-350 of SEQ ID NO: 69, at most 85% amino acid identity withamino acids 247-350 of SEQ ID NO: 69, at most 90% amino acid identitywith amino acids 247-350 of SEQ ID NO: 69 or at most 95% amino acididentity with amino acids 247-350 of SEQ ID NO: 69.

In other aspects of this embodiment, an Activin E comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid substitutionsrelative to amino acids 247-350 of SEQ ID NO: 69. In other aspects ofthis embodiment, an Activin E comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 247-350of SEQ ID NO: 69. In yet other aspects of this embodiment, an Activin Ecomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 247-350 of SEQ ID NO: 69. In otheraspects of this embodiment, an Activin E comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids247-350 of SEQ ID NO: 69. In still other aspects of this embodiment, anActivin E comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 247-350 of SEQ ID NO: 69. In otheraspects of this embodiment, an Activin E comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids247-350 of SEQ ID NO: 69.

In other aspects of this embodiment, an Activin E comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to amino acids 247-350 of SEQ ID NO: 69. In other aspects ofthis embodiment, an Activin E comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 247-350 ofSEQ ID NO: 69. In yet other aspects of this embodiment, an Activin Ecomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 247-350 of SEQ ID NO: 69. In other aspects ofthis embodiment, an Activin E comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 247-350 of SEQID NO: 69. In still other aspects of this embodiment, an Activin Ecomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 247-350 of SEQ ID NO: 69. In other aspects ofthis embodiment, an Activin E comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 247-350 of SEQID NO: 69.

In another embodiment, an enhanced binding domain derived from anInhibin A. In another embodiment, an enhanced binding domain derivedfrom an Inhibin A of SEQ ID NO: 70. In an aspect of this embodiment, anenhanced binding domain derived from an Inhibin A comprises amino acids262-366 or amino acids 233-366 of SEQ ID NO: 70.

In other aspects of this embodiment, an Inhibin A comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 262-366 or amino acids 233-366 of SEQ ID NO: 70, at least 75%amino acid identity with amino acids 262-366 or amino acids 233-366 ofSEQ ID NO: 70, at least 80% amino acid identity with amino acids 262-366or amino acids 233-366 of SEQ ID NO: 70, at least 85% amino acididentity with amino acids 262-366 or amino acids 233-366 of SEQ ID NO:70, at least 90% amino acid identity with amino acids 262-366 or aminoacids 233-366 of SEQ ID NO: 70 or at least 95% amino acid identity withamino acids 262-366 or amino acids 233-366 of SEQ ID NO: 70. In yetother aspects of this embodiment, an Inhibin A comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 262-366or amino acids 233-366 of SEQ ID NO: 70, at most 75% amino acid identitywith amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 70, atmost 80% amino acid identity with amino acids 262-366 or amino acids233-366 of SEQ ID NO: 70, at most 85% amino acid identity with aminoacids 262-366 or amino acids 233-366 of SEQ ID NO: 70, at most 90% aminoacid identity with amino acids 262-366 or amino acids 233-366 of SEQ IDNO: 70 or at most 95% amino acid identity with amino acids 262-366 oramino acids 233-366 of SEQ ID NO: 70.

In other aspects of this embodiment, an Inhibin A comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid substitutionsrelative to amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 70.In other aspects of this embodiment, an Inhibin A comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid substitutionsrelative to amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 70.In yet other aspects of this embodiment, an Inhibin A comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 70.In other aspects of this embodiment, an Inhibin A comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 70.In still other aspects of this embodiment, an Inhibin A comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 70.In other aspects of this embodiment, an Inhibin A comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 70.

In other aspects of this embodiment, an Inhibin A comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 70.In other aspects of this embodiment, an Inhibin A comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to amino acids 262-366 or amino acids 233-366 of SEQ ID NO: 70.In yet other aspects of this embodiment, an Inhibin A comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 262-366 or amino acids 233-366 of SEQ ID NO: 70. In otheraspects of this embodiment, an Inhibin A comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 262-366 oramino acids 233-366 of SEQ ID NO: 70. In still other aspects of thisembodiment, an Inhibin A comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 262-366 or aminoacids 233-366 of SEQ ID NO: 70. In other aspects of this embodiment, anInhibin A comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid additions relative to amino acids 262-366 or amino acids 233-366 ofSEQ ID NO: 70.

An enhanced targeting domain disclosed in the present specificationreplaces the binding activity of the Clostridial toxin binding domainfound in naturally occurring Clostridial toxins. As used herein, theterm “Clostridial toxin binding domain” is synonymous with “Clostridialtoxin H_(C) region” and means any naturally occurring Clostridial toxinpolypeptide that can execute the cell binding step of the intoxicationprocess, including, e.g., the binding of the Clostridial toxin to aClostridial toxin-specific receptor system located on the plasmamembrane surface of a target cell. It is envisioned that replacement ofthe binding activity can be achieved by, e.g., replacing the entireClostridial toxin binding domain with an enhanced targeting domain;replacing a portion of a Clostridial toxin binding domain with anenhanced targeting domain, with the proviso that the portion of aClostridial toxin binding domain remaining cannot selectively bind toits Clostridial toxin receptor system; and operationally-linking anenhanced targeting domain to a Clostridial toxin comprising aClostridial toxin binding domain, with the proviso that the aClostridial toxin binding domain is altered so that it cannotselectively bind to its Clostridial toxin receptor system.

The three-dimensional crystal structures of BoNT/A, BoNT/B and the H_(C)domain of TeNT indicate that the three functional domains of Clostridialneurotoxins are structurally distinct. The HEXXH consensus motif of thelight chain forms the tetrahedral zinc binding pocket of the catalyticsite located in a deep cleft on the protein surface that is accessibleby a channel. The structure of the H_(N) and H_(C) domains consistsprimarily of β-sheet topologies that are linked by a single α-helix. Thecylindrical-shaped H_(N) domain comprises two long amphipathic α-helicesthat resemble the coiled-coil motif found in some viral proteins. TheH_(N) domain also forms a long unstructured loop called the‘translocation belt,’ which wraps around a large negatively chargedcleft of the light chain that blocks access of the zinc atom to thecatalytic-binding pocket of active site. The H_(C) domain comprises twodistinct structural features of roughly equal size that indicatefunction. The first, designated the H_(CN) subdomain, is located in theamino half of the H_(C) domain. The H_(CN) subdomain forms a β-barrel,jelly-roll fold. The H_(CC) subdomain is the second subdomain thatcomprises the H_(C) domain. This carboxy-terminal subdomain comprises amodified β-trefoil domain which forms three distinct carbohydratebinding regions that resembles the carbohydrate binding moiety found inmany sugar-binding proteins, such as, e.g., serum amyloid P, sialidase,cryia, insecticidal α-endotoxin and lectins. Biochemical studiesindicate that the β-trefoil domain structure of the H_(CC) subdomainappears to mediate the binding to specific carbohydrate containingcomponents of the Clostridial toxin receptor system on the cell surface,see, e.g., Krzysztof Ginalski et al., Structure-based Sequence Alignmentfor the Beta-Trefoil Subdomain of the Clostridial Neurotoxin FamilyProvides Residue Level Information About the Putative GangliosideBinding Site, 482(1-2) FEBS Lett. 119-124 (2000). The H_(C) domain tiltsaway from the H_(N) domain exposing the surface loops and making themaccessible for binding. No contacts occur between the light chain andthe H_(C) domain.

Proteins containing the structural β-trefoil domain represents a diversegroup of proteins, see, e.g., C. A. Orengo et al., Protein Superfamiliesand Domain Superfolds, 372 Nature 631-634 (1994). The β-trefoil domaincomprises a six-stranded β-barrel closed off at one end by threeβ-hairpin structures that exhibits a characteristic pseudo-threefoldaxis symmetry. The monomeric structural unit of this three-fold symmetryis referred to as the β-trefoil fold that contains four β-sheetsorganized as a pair of antiparallel β-sheets. Dividing each of theseβ-trefoil folds is a β-hairpin turn. Therefore, in a linear fashion, aβ-trefoil domain comprises four β-sheets of the first β-trefoil fold, aβ-hairpin turn, four β-sheets of the second β-trefoil fold, a secondβ-hairpin turn four β-sheets of the third β-trefoil fold. Because thefirst hairpin turn is located between the fourth and fifth β-sheets ofthe β-trefoil domain, it is designated the β4/β5 β-hairpin turn.Likewise, since the second hairpin turn is located between the eight andninth β-sheets of the β-trefoil domain, it is designated the β8/β9β-hairpin turn.

As is typical for proteins containing a β-trefoil fold, the overallamino acid sequence identity of the H_(CC) subdomain between Clostridialtoxins is low. However, key residues essential for binding activity havebeen identified by structural analysis and mutagenesis experiments, see,e.g., Krzysztof Ginalski et al., Structure-based Sequence Alignment forthe Beta-Trefoil Subdomain of the Clostridial Neurotoxin Family ProvidesResidue Level Information About the Putative Ganglioside Binding Site,482(1-2) FEBS Lett. 119-124 (2000); and Andreas Rummel et al., The H_(CC)-Domain of Botulinum Neurotoxins A and B Exhibits a SingularGanglioside Binding Site Displaying Serotype Specific CarbohydrateInteraction, 51(3) Mol. Microbiol. 631-643 (2004). Additionally,research has elucidated that β4/β5 and β8/β9 β-hairpin turns areimportant in conferring the proper pseudo-threefold axis symmetryobserved in the β-trefoil domain and that these turns are important forβ-trefoil domain stability, see, e.g., Stephen R. Brych et al.,Structure and Stability Effects of Mutations Designed to Increase thePrimary Sequence Symmetry Within the Core Region of a β-trefoil, 10Protein Sci. 2587-2599 (2001); Jaewon Kim et al., Alternative Type I andI′ Turn Conformations in the β8/β9 β-hairpin of Human Acidic FibroblastGrowth Factor, 11 Protein Sci. 459-466 (2002); Jaewon Kim et al.,Sequence swapping Does Not Result in Conformation Swapping for the β4/β5and β8/β9 β-hairpin Turns in Human Acidic Fibroblast Growth Factor, 14Protein Sci. 351-359 (2005). The amino acid sequences comprising theβ-trefoil domains found in various Clostridial toxins are shown in Table2.

TABLE 2 β-trefoil Domains of Clostridial Toxins Amino Acid SequenceRegion of Carbohydrate Binding Moieties β4/β5 β8/β9 Protein SEQ ID NO:α-fold β-hairpin turn β-fold β-hairpin turn γ-fold BoNT/A 1 1111-11621163-1178 1179-1223 1224-1236 1237-1296 BoNT/B 2 1098-1147 1148-11651166-1210 1211-1222 1223-1291 BoNT/C1 3 1112-1150 1151-1166 1167-12181219-1229 1230-1291 BoNT/D 4 1099-1137 1138-1153 1154-1207 1208-12181219-1276 BoNT/E 5 1086-1129 1130-1146 1147-1190 1191-1198 1199-1252BoNT/F 6 1106-1152 1153-1171 1172-1213 1214-1221 1222-1274 BoNT/G 71106-1153 1154-1172 1173-1218 1219-1230 1231-1297 TeNT 8 1128-11771178-1194 1195-1240 1241-1254 1255-1315

Thus, in one embodiment, a Clostridial toxin binding domain comprisingan H_(C) region can be replaced with an enhance binding domain disclosedin the present specification. In aspects of this embodiment, a BoNT/AH_(C) region can be replaced with an enhanced targeting domain, a BoNT/BH_(C) region can be replaced with an enhanced targeting domain, aBoNT/C1H_(C) region can be replaced with an enhanced targeting domain, aBoNT/D H_(C) region can be replaced with an enhanced targeting domain, aBoNT/E H_(C) region can be replaced with an enhanced targeting domain, aBoNT/F H_(C) region can be replaced with an enhanced targeting domain, aBoNT/G H_(C) region can be replaced with an enhanced targeting domainand a TeNT H_(C) region can be replaced with an enhanced targetingdomain. In other aspects of this embodiment, a BoNT/A H_(C) regioncomprising amino acids 872-1296 of SEQ ID NO: 1 can be replaced with anenhanced targeting domain, a BoNT/B H_(C) region comprising amino acids859-1291 of SEQ ID NO: 2 can be replaced with an enhanced targetingdomain, a BoNT/C1H_(C) region comprising amino acids 867-1291 of SEQ IDNO: 3 can be replaced with an enhanced targeting domain, a BoNT/D H_(C)region comprising amino acids 863-1276 of SEQ ID NO: 4 can be replacedwith an enhanced targeting domain, a BoNT/E H_(C) region comprisingamino acids 846-1252 of SEQ ID NO: 5 can be replaced with an enhancedtargeting domain, a BoNT/F H_(C) region comprising amino acids 865-1274of SEQ ID NO: 6 can be replaced with an enhanced targeting domain, aBoNT/G H_(C) region comprising amino acids 864-1297 of SEQ ID NO: 7 canbe replaced with an enhanced targeting domain and a TeNT H_(C) regioncomprising amino acids 880-1315 of SEQ ID NO: 8 can be replaced with anenhanced targeting domain.

In another embodiment, a Clostridial toxin binding domain comprising anH_(CC) region can be replaced with an enhance binding domain disclosedin the present specification. In aspects of this embodiment, a BoNT/AH_(CC) region can be replaced with an enhanced targeting domain, aBoNT/B H_(CC) region can be replaced with an enhanced targeting domain,a BoNT/C1H_(CC) region can be replaced with an enhanced targetingdomain, a BoNT/D H_(CC) region can be replaced with an enhancedtargeting domain, a BoNT/E H_(CC) region can be replaced with anenhanced targeting domain, a BoNT/F H_(CC) region can be replaced withan enhanced targeting domain, a BoNT/G H_(CC) region can be replacedwith an enhanced targeting domain and a TeNT H_(CC) region can bereplaced with an enhanced targeting domain. In other aspects of thisembodiment, a BoNT/A H_(CC) region comprising amino acids 1092-1296 ofSEQ ID NO: 1 can be replaced with an enhanced targeting domain, a BoNT/BH_(CC) region comprising amino acids 1079-1291 of SEQ ID NO: 2 can bereplaced with an enhanced targeting domain, a BoNT/C1H_(CC) regioncomprising amino acids 1093-1291 of SEQ ID NO: 3 can be replaced with anenhanced targeting domain, a BoNT/D H_(CC) region comprising amino acids1080-1276 of SEQ ID NO: 4 can be replaced with an enhanced targetingdomain, a BoNT/E H_(CC) region comprising amino acids 1067-1252 of SEQID NO: 5 can be replaced with an enhanced targeting domain, a BoNT/FH_(CC) region comprising amino acids 1087-1274 of SEQ ID NO: 6 can bereplaced with an enhanced targeting domain, a BoNT/G H_(CC) regioncomprising amino acids 1087-1297 of SEQ ID NO: 7 can be replaced with anenhanced targeting domain and a TeNT H_(CC) region comprising aminoacids 1109-1315 of SEQ ID NO: 8 can be replaced with an enhancedtargeting domain.

In another embodiment, an enhance binding domain disclosed in thepresent specification is operationally-linked to a Clostridial toxincomprising a Clostridial toxin binding domain altered so that it cannotselectively bind to its Clostridial toxin receptor system. As usedherein, the term “altered,” when referring to a Clostridial toxinbinding domain, means a naturally occurring Clostridial toxin bindingdomain modified to eliminate or reduce the binding activity of theClostridial toxin binding domain so that the domain can no longerselectively bind to a Clostridial toxin receptor system. By definition,an altered Clostridial toxin binding domain has at least one amino acidchange from the corresponding region of the disclosed referencesequences (see Table 1) and can be described in percent identity to thecorresponding region of that reference sequence. As non-limitingexamples, a modified BoNT/A H_(C) region comprising amino acids 872-1296of SEQ ID NO: 1 will have at least one amino acid difference, such as,e.g., an amino acid substitution, deletion or addition, as compared tothe amino acid region 872-1296 of SEQ ID NO: 1; a modified BoNT/B H_(C)region comprising amino acids 859-1291 of SEQ ID NO: 2 will have atleast one amino acid difference, such as, e.g., an amino acidsubstitution, deletion or addition, as compared to the amino acid region859-1291 of SEQ ID NO: 2; a modified BoNT/C1H_(C) region comprisingamino acids 867-1291 of SEQ ID NO: 3 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the amino acid region 867-1291 of SEQ ID NO: 3;a modified BoNT/D H_(C) region comprising amino acids 863-1276 of SEQ IDNO: 4 will have at least one amino acid difference, such as, e.g., anamino acid substitution, deletion or addition, as compared to the aminoacid region 863-1276 of SEQ ID NO: 4; a modified BoNT/E H_(C) regioncomprising amino acids 846-1252 of SEQ ID NO: 5 will have at least oneamino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to the amino acid region 846-1252 ofSEQ ID NO: 5; a modified BoNT/F H_(C) region comprising amino acids865-1274 of SEQ ID NO: 6 will have at least one amino acid difference,such as, e.g., an amino acid substitution, deletion or addition, ascompared to the amino acid region 865-1274 of SEQ ID NO: 6; a modifiedBoNT/G H_(C) region comprising amino acids 864-1297 of SEQ ID NO: 7 willhave at least one amino acid difference, such as, e.g., an amino acidsubstitution, deletion or addition, as compared to the amino acid region864-1297 of SEQ ID NO: 7; and a modified TeNT H_(C) region comprisingamino acids 880-1315 of SEQ ID NO: 8 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the amino acid region 880-1315 of SEQ ID NO: 8.

In aspects of this embodiment, an altered Clostridial toxin H_(C) regioncomprises a polypeptide having, e.g., at least 70% amino acid identitywith its reference sequence, at least 75% amino acid identity with itsreference sequence, at least 80% amino acid identity with its referencesequence, at least 85% amino acid identity with its reference sequence,at least 90% amino acid identity with its reference sequence or at least95% amino acid identity with its reference sequence. In yet otheraspects of this embodiment, an altered Clostridial toxin H_(C) regioncomprises a polypeptide having, e.g., at most 70% amino acid identitywith its reference sequence, at most 75% amino acid identity with itsreference sequence, at most 80% amino acid identity with its referencesequence, at most 85% amino acid identity with its reference sequence,at most 90% amino acid identity with its reference sequence or at most95% amino acid identity with its reference sequence.

In other aspects of this embodiment, an altered Clostridial toxin H_(C)region comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200non-contiguous amino acid substitutions relative to its referencesequence. In other aspects of this embodiment, an altered Clostridialtoxin H_(C) region comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 non-contiguous amino acid substitutions relative to its referencesequence. In yet other aspects of this embodiment, an alteredClostridial toxin H_(C) region comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 non-contiguous amino acid deletions relative to itsreference sequence. In other aspects of this embodiment, an alteredClostridial toxin H_(C) region comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 non-contiguous amino acid deletions relative to itsreference sequence. In still other aspects of this embodiment, analtered Clostridial toxin H_(C) region comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relativeto its reference sequence. In other aspects of this embodiment, analtered Clostridial toxin H_(C) region comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relativeto its reference sequence.

In other aspects of this embodiment, an altered Clostridial toxin H_(C)region comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid substitutions relative to its reference sequence.In other aspects of this embodiment, an altered Clostridial toxin H_(C)region comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid substitutions relative to its reference sequence.In yet other aspects of this embodiment, an altered Clostridial toxinH_(C) region comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or200 contiguous amino acid deletions relative to its reference sequence.In other aspects of this embodiment, an altered Clostridial toxin H_(C)region comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid deletions relative to its reference sequence. Instill other aspects of this embodiment, an altered Clostridial toxinH_(C) region comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or200 contiguous amino acid additions relative to its reference sequence.In other aspects of this embodiment, an altered Clostridial toxin H_(C)region comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid additions relative to its reference sequence.

In another aspect of the invention, a modified Clostridial toxin withenhanced binding activity comprises, in part, a protease cleavage siteis located within a di-chain loop region. As used herein, the term“di-chain loop region” means the amino acid sequence of a Clostridialtoxin containing a protease cleavage site used to convert thesingle-chain form of a Clostridial toxin into the di-chain form.Non-limiting examples of a Clostridial toxin di-chain loop region,include, a di-chain loop region of BoNT/A comprising amino acids 430-454of SEQ ID NO: 1; a di-chain loop region of BoNT/B comprising amino acids437-446 of SEQ ID NO: 2; a di-chain loop region of BoNT/C1 comprisingamino acids 437-453 of SEQ ID NO: 3; a di-chain loop region of BoNT/Dcomprising amino acids 437-450 of SEQ ID NO: 4; a di-chain loop regionof BoNT/E comprising amino acids 412-426 of SEQ ID NO: 5; a di-chainloop region of BoNT/F comprising amino acids 429-445 of SEQ ID NO: 6; adi-chain loop region of BoNT/G comprising amino acids 436-450 of SEQ IDNO: 7; and a di-chain loop region of TeNT comprising amino acids 439-467of SEQ ID NO: 8 (Table 8).

TABLE 8 Di-chain Loop Region of Clostridial Toxins SEQDi-chain Loop Region Containing the ID Light ChainNaturally-occurring Protease Cleavage Heavy Chain Toxin NO: Region SiteRegion BoNT/A 1 NMNFTKLKNFTGLFEFYKLL CVRGIITSKTKSLDKGYNK*----ALNDLCIKVNNWDL BoNT/B 2 KQAYEEISKEHLAVYKIQM CKSVK*-------------------APGICIDVDNEDL BoNT/C1 3 PALRKVNPENMLYLFTKF CHKAIDGRSLYNK*------------TLDCRELLVKNTDL BoNT/D 4 PALQKLSSESVVDLFTKV CLRLTKNSR*---------------DDSTCIKVKNNRL BoNT/E 5 IITPITGRGLVKKIIRF CKNIVSVKGIR*--------------KSICIEINNGEL BoNT/F 6 IIDSIPDKGLVEKIVKF CKSVIPRKGTK*------------APPRLCIRVNNSEL BoNT/G 7 KEAYEEISLEHLVIYRIAM CKPVMYKNTGK*--------------SEQCIIVNNEDL TeNT 8 TNAFRNVDGSGLVSKLIGL CKKIIPPTNIRENLYNRTA*SLTDLGGELCIKIKNEDL The amino acid sequence displayed are as follows: BoNT/A,residues 325-462 of SEQ ID No: 1; BoNT/B, residues 332-454 of SEQ ID No:2; BoNT/C1, residues 334-463 of SEQ ID No: 3; BoNT/D, residues 334-458of SEQ ID No: 4; BoNT/E, residues 311-434 of SEQ ID No: 5; BoNT/F,residues 328-453 of SEQ ID No: 6; BoNT/G, residues 331-458 of SEQ ID No:7; and TeNT, residues 334-474 of SEQ ID No: 8. An asterisks (*)indicates the peptide bond that is cleaved by a Clostridial toxinprotease.

In is envisioned that any and all protease cleavage sites can be used toconvert the single-chain polypeptide form of a Clostridial toxin intothe di-chain form, including, without limitation, endogenous di-chainloop protease cleavage sites and exogenous protease cleavage sites.

As used herein, the term “endogenous di-chain loop protease cleavagesite” is synonymous with a “naturally occurring di-chain loop proteasecleavage site” and means a naturally occurring protease cleavage sitefound within the di-chain loop region of a naturally occurringClostridial toxin and includes, without limitation, naturally occurringClostridial toxin di-chain loop protease cleavage site variants, suchas, e.g., Clostridial toxin di-chain loop protease cleavage siteisoforms and Clostridial toxin di-chain loop protease cleavage sitesubtypes. Non-limiting examples of an endogenous protease cleavage site,include, e.g., a BoNT/A di-chain loop protease cleavage site, a BoNT/Bdi-chain loop protease cleavage site, a BoNT/C1 di-chain loop proteasecleavage site, a BoNT/D di-chain loop protease cleavage site, a BoNT/Edi-chain loop protease cleavage site, a BoNT/F di-chain loop proteasecleavage site, a BoNT/G di-chain loop protease cleavage site and a TeNTdi-chain loop protease cleavage site.

As mentioned above, Clostridial toxins are translated as a single-chainpolypeptide of approximately 150 kDa that is subsequently cleaved byproteolytic scission within a disulfide loop by a naturally-occurringprotease. This posttranslational processing yields a di-chain moleculecomprising an approximately 50 kDa light chain (LC) and an approximately100 kDa heavy chain (HC) held together by a single disulphide bond andnoncovalent interactions. While the identity of the protease iscurrently unknown, the di-chain loop protease cleavage site for manyClostridial toxins has been determined. In BoNTs, cleavage at K448-A449converts the single polypeptide form of BoNT/A into the di-chain form;cleavage at K441-A442 converts the single polypeptide form of BoNT/Binto the di-chain form; cleavage at K449-T450 converts the singlepolypeptide form of BoNT/C1 into the di-chain form; cleavage atR445-D446 converts the single polypeptide form of BoNT/D into thedi-chain form; cleavage at R422-K423 converts the single polypeptideform of BoNT/E into the di-chain form; cleavage at K439-A440 convertsthe single polypeptide form of BoNT/F into the di-chain form; andcleavage at K446-S447 converts the single polypeptide form of BoNT/Ginto the di-chain form. Proteolytic cleavage of the single polypeptideform of TeNT at A457-S458 results in the di-chain form. Such a di-chainloop protease cleavage site is operably-linked in-frame to a modifiedClostridial toxin as a fusion protein. However, it should also be notedthat additional cleavage sites within the di-chain loop also appear tobe cleaved resulting in the generation of a small peptide fragment beinglost. As a non-limiting example, BoNT/A single-chain polypeptide cleaveultimately results in the loss of a ten amino acid fragment within thedi-chain loop.

Thus, in an embodiment, a protease cleavage site comprising anendogenous Clostridial toxin di-chain loop protease cleavage site isused to convert the single-chain toxin into the di-chain form. Inaspects of this embodiment, conversion into the di-chain form byproteolytic cleavage occurs from a site comprising, e.g., a BoNT/Adi-chain loop protease cleavage site, a BoNT/B di-chain loop proteasecleavage site, a BoNT/C1 di-chain loop protease cleavage site, a BoNT/Ddi-chain loop protease cleavage site, a BoNT/E di-chain loop proteasecleavage site, a BoNT/F di-chain loop protease cleavage site, a BoNT/Gdi-chain loop protease cleavage site or a TeNT di-chain loop proteasecleavage site.

In other aspects of this embodiment, conversion into the di-chain formby proteolytic cleavage occurs from a site comprising, e.g., a di-chainloop region of BoNT/A comprising amino acids 430-454 of SEQ ID NO: 1; adi-chain loop region of BoNT/B comprising amino acids 437-446 of SEQ IDNO: 2; a di-chain loop region of BoNT/C1 comprising amino acids 437-453of SEQ ID NO: 3; a di-chain loop region of BoNT/D comprising amino acids437-450 of SEQ ID NO: 4; a di-chain loop region of BoNT/E comprisingamino acids 412-426 of SEQ ID NO: 5; a di-chain loop region of BoNT/Fcomprising amino acids 429-445 of SEQ ID NO: 6; a di-chain loop regionof BoNT/G comprising amino acids 436-450 of SEQ ID NO: 7; or a di-chainloop region of TeNT comprising amino acids 439-467 of SEQ ID NO: 8.

It is also envisioned that an exogenous protease cleavage site can beused to convert the single-chain polypeptide form of a modifiedClostridial toxin disclosed in the present specification into thedi-chain form. As used herein, the term “exogenous protease cleavagesite” is synonymous with a “non-naturally occurring protease cleavagesite” and means a protease cleavage site that is not normally present ina di-chain loop region from a naturally occurring Clostridial toxin.Non-limiting examples of exogenous protease cleavage sites include,e.g., an enterokinase cleavage site (Table 5); a Thrombin cleavage site(Table 9); a Factor Xa cleavage site (Table 9); a human rhinovirus 3Cprotease cleavage site (Table 9); a tobacco etch virus (TEV) proteasecleavage site (Table 9); a dipeptidyl aminopeptidase cleavage site; asmall ubiquitin-like modifier (SUMO)/ubiquitin-like protein-1 (ULP-1)protease cleavage site, such as, e.g.,MADSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG (SEQ ID. NO: 57); and aClostridial toxin substrate cleavage site.

TABLE 9 Exogenous Protease Clevage Sites Protease Cleavage Non-limitingSEQ ID Site Consensus Sequence Examples NO: Bovine enterokinase DDDDK*DDDDK* 71 Tobacco Etch Virus E P⁵ P⁴YP²Q*(G/S), ENLYFQ*G 72 (TEV)where P², P⁴ and P⁵ can be any amino acid ENLYFQ*S 73 ENIYTQ*G 74ENIYTQ*S 75 ENIYLQ*G 76 ENIYLQ*S 77 ENVYFQ*G 78 ENVYSQ*S 79 ENVYSQ*G 80ENVYSQ*S 81 Human Rhinovirus 3C P⁵P⁴LFQ*GP EALFQ*GP 82where P⁴ is G, A, V, L, I, M, S or T and P⁵ can any EVLFQ*GP 83amino acid, with D or E preferred. ELLFQ*GP 84 DALFQ*GP 85 DVLFQ*GP 86DLLFQ*GP 87 SUMO/ULP-1 Tertiary structure polypeptide-G* 88 ThrombinP³P²(R/K)*P^(1′), GVR*G 89where P³ is any amino acid and P² or P^(1′) is G with SAR*G 90the other position being any amino acid SLR*G 91 DGR*I 92 QGK*I 93Thrombin P⁴P³P(R/K)*P^(1′)P^(2′) LVPR*GS 94where P^(1′) and P^(2′) can be any amino acid except for LVPK*GS 95acidic amino acids like D or E; and P³ and P⁴ are FIPR*TF 96hydrophobic amino acids like F, L, I, Y, W, V, M, VLPR*SF 97 P, C or AIVPR*SF 98 IVPR*GY 99 VVPR*GV 100 VLPR*LI 101 VMPR*SL 102 MFPR*SL 103Coagulation Factor Xa I(E/D)GR* IDGR* 104 IEGR* 105 An asterisks (*)indicates the peptide bond that is cleaved by the indicated protease.

As mentioned above, a Clostridial toxin is converted from a singlepolypeptide form into a di-chain molecule by proteolytic cleavage. Whilethe naturally-occurring protease is currently not known, cleavage occurswithin the di-chain loop region between the two cysteine residues thatform the disulfide bridge (see Table 8). Replacement of an endogenousprotease cleavage site with an exogenous protease cleavage site willenable cleavage of a modified Clostridial toxin disclosed in the presentspecification when expressed in an organism that does not produce thenaturally-occurring protease used to cleave the di-chain loop region ofa toxin. Similarly, an addition of an exogenous protease cleavage sitein the di-chain loop region will also enable cleavage of a modifiedClostridial toxin disclosed in the present specification when expressedin an organism that does not produce the naturally-occurring proteaseused to cleave the di-chain loop region of a toxin.

It is envisioned that an exogenous protease cleavage site of any and alllengths can be useful in aspects of the present invention with theproviso that the exogenous protease cleavage site is capable of beingcleaved by its respective protease. Thus, in aspects of this embodiment,an exogenous protease cleavage site can be, e.g., at least 6 amino acidsin length, at least 7 amino acids in length, at least 8 amino acids inlength, at least 9 amino acids in length, at least 10 amino acids inlength, at least 15 amino acids in length, at least 20 amino acids inlength, at least 25 amino acids in length, at least 30 amino acids inlength, at least 40 amino acids in length, at least 50 amino acids inlength or at least 60 amino acids in length. In other aspects of thisembodiment, an exogenous protease cleavage site can be, e.g., at most 6amino acids in length, at most 7 amino acids in length, at most 8 aminoacids in length, at most 9 amino acids in length, at most 10 amino acidsin length, at most 15 amino acids in length, at most 20 amino acids inlength, at most 25 amino acids in length, at most 30 amino acids inlength, at most 40 amino acids in length, at most 50 amino acids inlength or at most 60 amino acids in length.

In aspects of this embodiment, a di-chain loop region can be modified tosubstitute a naturally-occurring protease cleavage site for an exogenousprotease cleavage site. In this type of modification, thenaturally-occurring protease cleavage site is made inoperable and thuscan not be cleaved by its protease. Only the exogenous protease cleavagesite can be cleaved by its corresponding exogenous protease. In thistype of modification, the exogenous protease site is operably-linkedin-frame to a modified Clostridial toxin as a fusion protein and thesite can be cleaved by its respective exogenous protease. As anon-limiting example, a single-chain modified BoNT/A comprising anexogenous protease cleavage site in the di-chain loop region can becleaved by its respective exogenous protease to produce the di-chainform of the toxin.

In other aspects of this embodiment, a di-chain loop region can bemodified to include an exogenous protease cleavage site in addition tothe naturally-occurring protease cleavage site. In this type ofmodification, both cleavage sites are operably-linked in-frame to amodified Clostridial toxin as a fusion protein and both sites can becleaved by their respective proteases. As a non-limiting example, asingle-chain modified BoNT/A that comprises a di-chain loop containingboth the naturally-occurring BoNT/A di-chain loop protease cleavage siteand an exogenous protease cleavage site can be cleaved by either thenaturally occurring di-chain loop protease or by the appropriateexogenous protease to produce the di-chain form of the toxin.

A naturally-occurring protease cleavage site can be made inoperable byaltering at least the two amino acids flanking the peptide bond cleavedby the naturally-occurring di-chain loop protease. More extensivealterations can be made, with the proviso that the two cysteine residuesof the di-chain loop region remain intact and can still form thedisulfide bridge. Non-limiting examples of an amino acid alterationinclude deletion of an amino acid or replacement of the original aminoacid with a different amino acid. Thus, in one embodiment, anaturally-occurring protease cleavage site is made inoperable byaltering the two amino acids flanking the peptide bond cleaved by anaturally-occurring protease. In other aspects of this embodiment, anaturally-occurring protease cleavage site is made inoperable byaltering, e.g., at least three amino acids including the two amino acidsflanking the peptide bond cleaved by a naturally-occurring protease; atleast four amino acids including the two amino acids flanking thepeptide bond cleaved by a naturally-occurring protease; at least fiveamino acids including the two amino acids flanking the peptide bondcleaved by a naturally-occurring protease; at least six amino acidsincluding the two amino acids flanking the peptide bond cleaved by anaturally-occurring protease; at least seven amino acids including thetwo amino acids flanking the peptide bond cleaved by anaturally-occurring protease; at least eight amino acids including thetwo amino acids flanking the peptide bond cleaved by anaturally-occurring protease; at least nine amino acids including thetwo amino acids flanking the peptide bond cleaved by anaturally-occurring protease; at least ten amino acids including the twoamino acids flanking the peptide bond cleaved by a naturally-occurringprotease; at least 15 amino acids including the two amino acids flankingthe peptide bond cleaved by a naturally-occurring protease; or at least20 amino acids including the two amino acids flanking the peptide bondcleaved by a naturally-occurring protease.

In still other aspects of this embodiment, a naturally-occurringdi-chain protease cleavage site is made inoperable by altering, e.g., atmost three amino acids including the two amino acids flanking thepeptide bond cleaved by a naturally-occurring protease; at most fouramino acids including the two amino acids flanking the peptide bondcleaved by a naturally-occurring protease; at most five amino acidsincluding the two amino acids flanking the peptide bond cleaved by anaturally-occurring protease; at most six amino acids including the twoamino acids flanking the peptide bond cleaved by a naturally-occurringprotease; at most seven amino acids including the two amino acidsflanking the peptide bond cleaved by a naturally-occurring protease; atmost eight amino acids including the two amino acids flanking thepeptide bond cleaved by a naturally-occurring protease; at most nineamino acids including the two amino acids flanking the peptide bondcleaved by a naturally-occurring protease; at most ten amino acidsincluding the two amino acids flanking the peptide bond cleaved by anaturally-occurring protease; at most 15 amino acids including the twoamino acids flanking the peptide bond cleaved by a naturally-occurringprotease; or at most 20 amino acids including the two amino acidsflanking the peptide bond cleaved by a naturally-occurring protease.

In an embodiment, an exogenous protease cleavage site is located withinthe di-chain loop of a modified Clostridial toxin. In aspects of thisembodiment, a modified Clostridial toxin comprises an exogenous proteasecleavage site comprises, e.g., a bovine enterokinase protease cleavagesite, a Tobacco Etch Virus protease cleavage site, a Human Rhinovirus 3Cprotease cleavage site, a SUMO/ULP-1 protease cleavage site, a Thrombinprotease cleavage site or a Factor Xa protease cleavage site. In otheraspects of this embodiment, an exogenous protease cleavage site islocated within the di-chain loop of, e.g., a modified BoNT/A, a modifiedBoNT/B, a modified BoNT/C1, a modified BoNT/D, a modified BoNT/E, amodified BoNT/F, a modified BoNT/G or a modified TeNT.

In an aspect of this embodiment, an exogenous protease cleavage site canbe, e.g., a bovine enterokinase cleavage site is located within thedi-chain loop of a modified Clostridial toxin. In other aspects of theembodiment, an exogenous protease cleavage site can be, e.g., a bovineenterokinase protease cleavage site located within the di-chain loop ofa modified Clostridial toxin comprises SEQ ID NO: 71. Is still otheraspects of this embodiment, a bovine enterokinase protease cleavage siteis located within the di-chain loop of, e.g., a modified BoNT/A, amodified BoNT/B, a modified BoNT/C1, a modified BoNT/D, a modifiedBoNT/E, a modified BoNT/F, a modified BoNT/G or a modified TeNT.

In another aspect of this embodiment, an exogenous protease cleavagesite can be, e.g., a Tobacco Etch Virus protease cleavage site islocated within the di-chain loop of a modified Clostridial toxin. Inother aspects of the embodiment, an exogenous protease cleavage site canbe, e.g., a Tobacco Etch Virus protease cleavage site located within thedi-chain loop of a modified Clostridial toxin comprises SEQ ID NO: 72,SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO:77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80 or SEQ ID NO: 81. Isstill other aspects of this embodiment, a Tobacco Etch Virus proteasecleavage site is located within the di-chain loop of, e.g., a modifiedBoNT/A, a modified BoNT/B, a modified BoNT/C1, a modified BoNT/D, amodified BoNT/E, a modified BoNT/F, a modified BoNT/G or a modifiedTeNT.

In still another aspect of this embodiment, an exogenous proteasecleavage site can be, e.g., a Human Rhinovirus 3C protease cleavage siteis located within the di-chain loop of a modified Clostridial toxin. Inother aspects of the embodiment, an exogenous protease cleavage site canbe, e.g., a Human Rhinovirus 3C protease cleavage site located withinthe di-chain loop of a modified Clostridial toxin comprises SEQ ID NO:82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 or SEQ IDNO: 87. Is still other aspects of this embodiment, a Human Rhinovirus 3Cprotease cleavage site is located within the di-chain loop of, e.g., amodified BoNT/A, a modified BoNT/B, a modified BoNT/C1, a modifiedBoNT/D, a modified BoNT/E, a modified BoNT/F, a modified BoNT/G or amodified TeNT.

In yet another aspect of this embodiment, an exogenous protease cleavagesite can be, e.g., a SUMO/ULP-1 protease cleavage site is located withinthe di-chain loop of a modified Clostridial toxin. In other aspects ofthe embodiment, an exogenous protease cleavage site can be, e.g., aSUMO/ULP-1 protease cleavage site located within the di-chain loop of amodified Clostridial toxin comprises SEQ ID NO: 88. Is still otheraspects of this embodiment, a SUMO/ULP-1 protease cleavage site islocated within the di-chain loop of, e.g., a modified BoNT/A, a modifiedBoNT/B, a modified BoNT/C1, a modified BoNT/D, a modified BoNT/E, amodified BoNT/F, a modified BoNT/G or a modified TeNT.

In a further aspect of this embodiment, an exogenous protease cleavagesite can be, e.g., a Thrombin protease cleavage site is located withinthe di-chain loop of a modified Clostridial toxin. In other aspects ofthe embodiment, an exogenous protease cleavage site can be, e.g., aThrombin protease cleavage site located within the di-chain loop of amodified Clostridial toxin comprises SEQ ID NO: 89, SEQ ID NO: 90, SEQID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95,SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO:100, SEQ ID NO: 101, SEQ ID NO: 102 or SEQ ID NO: 103. Is still otheraspects of this embodiment, a Thrombin protease cleavage site is locatedwithin the di-chain loop of, e.g., a modified BoNT/A, a modified BoNT/B,a modified BoNT/C1, a modified BoNT/D, a modified BoNT/E, a modifiedBoNT/F, a modified BoNT/G or a modified TeNT.

In another aspect of this embodiment, an exogenous protease cleavagesite can be, e.g., a Coagulation Factor Xa protease cleavage site islocated within the di-chain loop of a modified Clostridial toxin. Inother aspects of the embodiment, an exogenous protease cleavage site canbe, e.g., a Coagulation Factor Xa protease cleavage site located withinthe di-chain loop of a modified Clostridial toxin comprises SEQ ID NO:104 or SEQ ID NO: 105. Is still other aspects of this embodiment, aCoagulation Factor Xa protease cleavage site is located within thedi-chain loop of, e.g., a modified BoNT/A, a modified BoNT/B, a modifiedBoNT/C1, a modified BoNT/D, a modified BoNT/E, a modified BoNT/F, amodified BoNT/G or a modified TeNT.

In another embodiment, an exogenous protease site comprises aClostridial toxin substrate cleavage site. As used herein, the term“Clostridial toxin substrate cleavage site” means a scissile bondtogether with adjacent or non-adjacent recognition elements, or both,sufficient for detectable proteolysis at the scissile bond by aClostridial toxin under conditions suitable for Clostridial toxinprotease activity. By definition, a Clostridial toxin substrate cleavagesite is susceptible to cleavage by at least one Clostridial toxin underconditions suitable for Clostridial toxin protease activity.Non-limiting examples of Clostridial toxin substrate cleavage site aredisclosed in, e.g., Steward, L. E. et al., Self-Activating ClostridialToxins, U.S. Patent Application 60/718,616 (Sep. 19, 2005).

It is understood that a modified Clostridial toxin disclosed in thepresent specification can optionally include one or more additionalcomponents. As a non-limiting example of an optional component, amodified Clostridial toxin can further comprise a flexible regioncomprising a flexible spacer. Non-limiting examples of a flexible spacerinclude, e.g., a G-spacer GGGGS (SEQ ID NO: 106) or an A-spacer EAAAK(SEQ ID NO: 107). A flexible region comprising flexible spacers can beused to adjust the length of a polypeptide region in order to optimize acharacteristic, attribute or property of a polypeptide. Such a flexibleregion is operably-linked in-frame to the modified Clostridial toxin asa fusion protein. As a non-limiting example, a polypeptide regioncomprising one or more flexible spacers in tandem can be use to betterexpose a protease cleavage site thereby facilitating cleavage of thatsite by a protease. As another non-limiting example, a polypeptideregion comprising one or more flexible spacers in tandem can be use tobetter present an enhanced targeting domain, thereby facilitating thebinding of that enhanced targeting domain to its receptor system.

Thus, in an embodiment, a modified Clostridial toxin disclosed in thepresent specification can further comprise a flexible region comprisinga flexible spacer. In another embodiment, a modified Clostridial toxindisclosed in the present specification can further comprise flexibleregion comprising a plurality of flexible spacers in tandem. In aspectsof this embodiment, a flexible region can comprise in tandem, e.g., atleast 1 G-spacer, at least 2 G-spacers, at least 3 G-spacers, at least 4G-spacers or at least 5 G-spacers. In other aspects of this embodiment,a flexible region can comprise in tandem, e.g., at most 1 G-spacer, atmost 2 G-spacers, at most 3 G-spacers, at most 4 G-spacers or at most 5G-spacers. In still other aspects of this embodiment, a flexible regioncan comprise in tandem, e.g., at least 1 A-spacer, at least 2 A-spacers,at least 3 A-spacers, at least 4 A-spacers or at least 5 A-spacers. Instill other aspects of this embodiment, a flexible region can comprisein tandem, e.g., at most 1 A-spacer, at most 2 A-spacers, at most 3A-spacers, at most 4 A-spacers or at most 5 A-spacers. In another aspectof this embodiment, a modified Clostridial toxin can comprise a flexibleregion comprising one or more copies of the same flexible spacers, oneor more copies of different flexible-spacer regions, or any combinationthereof.

In aspects of this embodiment, a modified Clostridial toxin comprising aflexible spacer can be, e.g., a modified BoNT/A, a modified BoNT/B, amodified BoNT/C1, a modified BoNT/D, a modified BoNT/E, a modifiedBoNT/F, a modified BoNT/G or a modified TeNT.

It is envisioned that a modified Clostridial toxin disclosed in thepresent specification can comprise a flexible spacer in any and alllocations with the proviso that modified Clostridial toxin is capable ofperforming the intoxication process. In aspects of this embodiment, aflexible spacer is positioned between, e.g., an enzymatic domain and atranslocation domain, an enzymatic domain and an enhanced targetingdomain, an enzymatic domain and a protease cleavage site. In otheraspects of this embodiment, a G-spacer is positioned between, e.g., anenzymatic domain and a translocation domain, an enzymatic domain and anenhanced targeting domain, an enzymatic domain and a protease cleavagesite. In other aspects of this embodiment, a A-spacer is positionedbetween, e.g., an enzymatic domain and a translocation domain, anenzymatic domain and an enhanced targeting domain, an enzymatic domainand a protease cleavage site.

In other aspects of this embodiment, a flexible spacer is positionedbetween, e.g., an enhanced targeting domain and a translocation domain,an enhanced targeting domain and an enzymatic domain, an enhancedtargeting domain and a protease cleavage site. In other aspects of thisembodiment, a G-spacer is positioned between, e.g., an enhancedtargeting domain and a translocation domain, an enhanced targetingdomain and an enzymatic domain, an enhanced targeting domain and aprotease cleavage site. In other aspects of this embodiment, a A-spaceris positioned between, e.g., an enhanced targeting domain and atranslocation domain, an enhanced targeting domain and an enzymaticdomain, an enhanced targeting domain and a protease cleavage site.

In yet other aspects of this embodiment, a flexible spacer is positionedbetween, e.g., a translocation domain and an enzymatic domain, antranslocation domain and an enhanced targeting domain, an translocationdomain and a protease cleavage site. In other aspects of thisembodiment, a G-spacer is positioned between, e.g., a translocationdomain and an enzymatic domain, an translocation domain and an enhancedtargeting domain, an translocation domain and a protease cleavage site.In other aspects of this embodiment, a A-spacer is positioned between,e.g., a translocation domain and an enzymatic domain, an translocationdomain and an enhanced targeting domain, a translocation domain and aprotease cleavage site.

As another non-limiting example of an optional component, a modifiedClostridial toxin can further comprise an epitope-binding region. Anepitope-binding region can be used in a wide variety of proceduresinvolving, e.g., protein purification and protein visualization. Such anepitope-binding region is operably-linked in-frame to a modifiedClostridial toxin as a fusion protein. Non-limiting examples of anepitope-binding region include, e.g., FLAG, Express™ (SEQ ID NO: 108),human Influenza virus hemaglutinin (HA) (SEQ ID NO: 109), humanp62^(c-Myc) protein (c-MYC) (SEQ ID NO: 110), Vesicular Stomatitis VirusGlycoprotein (VSV-G) (SEQ ID NO: 111), Substance P (SEQ ID NO: 112),glycoprotein-D precursor of Herpes simplex virus (HSV) (SEQ ID NO: 113),V5 (SEQ ID NO: 114), AU1 (SEQ ID NO: 115) and AU5 (SEQ ID NO: 116);affinity-binding, such as, e.g., polyhistidine (HIS) (SEQ ID NO: 117),streptavidin binding peptide (strep), and biotin or a biotinylationsequence; peptide-binding regions, such as, e.g., the glutathionebinding domain of glutathione-S-transferase, the calmodulin bindingdomain of the calmodulin binding protein, and the maltose binding domainof the maltose binding protein. Non-limiting examples of specificprotocols for selecting, making and using an appropriate binding peptideare described in, e.g., Epitope Tagging, pp. 17.90-17.93 (Sambrook andRussell, eds., Molecular Cloning A Laboratory Manual, Vol. 3, 3^(rd) ed.2001); Antibodies: A Laboratory Manual (Edward Harlow & David Lane,eds., Cold Spring Harbor Laboratory Press, 2^(nd) ed. 1998); and UsingAntibodies: A Laboratory Manual: Portable Protocol No. I (Edward Harlow& David Lane, Cold Spring Harbor Laboratory Press, 1998). In addition,non-limiting examples of binding peptides as well as well-characterizedreagents, conditions and protocols are readily available from commercialvendors that include, without limitation, BD Biosciences-Clontech, PaloAlto, Calif.; BD Biosciences Pharmingen, San Diego, Calif.; Invitrogen,Inc, Carlsbad, Calif.; QIAGEN, Inc., Valencia, Calif.; and Stratagene,La Jolla, Calif. These protocols are routine procedures well within thescope of one skilled in the art and from the teaching herein.

Thus, in an embodiment, a modified Clostridial toxin disclosed in thepresent specification can further comprise an epitope-binding region. Inanother embodiment, a modified Clostridial toxin disclosed in thepresent specification can further comprises a plurality ofepitope-binding regions. In aspects of this embodiment, a modifiedClostridial toxin can comprise, e.g., at least 1 epitope-binding region,at least 2 epitope-binding regions, at least 3 epitope-binding regions,at least 4 epitope-binding regions or at least 5 epitope-bindingregions. In other aspects of this embodiment, a modified Clostridialtoxin can comprise, e.g., at most 1 epitope-binding region, at most 2epitope-binding regions, at most 3 epitope-binding regions, at most 4epitope-binding regions or at most 5 epitope-binding regions. In anotheraspect of this embodiment, a modified Clostridial toxin can comprise oneor more copies of the same epitope-binding region, one or more copies ofdifferent epitope-binding regions, or any combination thereof.

The location of an epitope-binding region can be in various positions,including, without limitation, at the amino terminus of a modifiedClostridial toxin, within a modified Clostridial toxin, or at thecarboxyl terminus of a modified Clostridial toxin. Thus, in anembodiment, an epitope-binding region is located at the amino-terminusof a modified Clostridial toxin. In such a location, a start methionineshould be placed in front of the epitope-binding region. In addition, itis known in the art that when adding a polypeptide that isoperationally-linked to the amino terminus of another polypeptidecomprising the start methionine that the original methionine residue canbe deleted. This is due to the fact that the added polypeptide willcontain a new start methionine and that the original start methioninemay reduce optimal expression of the fusion protein. In aspects of thisembodiment, an epitope-binding region located at the amino-terminus of amodified Clostridial toxin disclosed in the present specification canbe, e.g., a FLAG, Express™ epitope-binding region, a human Influenzavirus hemaglutinin (HA) epitope-binding region, a human p62^(c-Myc)protein (c-MYC) epitope-binding region, a Vesicular Stomatitis VirusGlycoprotein (VSV-G) epitope-binding region, a Substance Pepitope-binding region, a glycoprotein-D precursor of Herpes simplexvirus (HSV) epitope-binding region, a V5 epitope-binding region, a AU1epitope-binding region, a AU5 epitope-binding region, a polyhistidineepitope-binding region, a streptavidin binding peptide epitope-bindingregion, a biotin epitope-binding region, a biotinylation epitope-bindingregion, a glutathione binding domain of glutathione-S-transferase, acalmodulin binding domain of the calmodulin binding protein or a maltosebinding domain of the maltose binding protein.

In another embodiment, an epitope-binding region is located at thecarboxyl-terminus of a modified Clostridial toxin. In aspects of thisembodiment, an epitope-binding region located at the carboxyl-terminusof a modified Clostridial toxin disclosed in the present specificationcan be, e.g., a FLAG, Express™ epitope-binding region, a human Influenzavirus hemaglutinin (HA) epitope-binding region, a human p62^(c-Myc)protein (c-MYC) epitope-binding region, a Vesicular Stomatitis VirusGlycoprotein (VSV-G) epitope-binding region, a Substance Pepitope-binding region, a glycoprotein-D precursor of Herpes simplexvirus (HSV) epitope-binding region, a V5 epitope-binding region, a AU1epitope-binding region, a AU5 epitope-binding region, a polyhistidineepitope-binding region, a streptavidin binding peptide epitope-bindingregion, a biotin epitope-binding region, a biotinylation epitope-bindingregion, a glutathione binding domain of glutathione-S-transferase, acalmodulin binding domain of the calmodulin binding protein or a maltosebinding domain of the maltose binding protein.

As another non-limiting example of an optional component, a modifiedClostridial toxin can further comprise an epitope-binding region. Anepitope-binding region can be used in a wide variety of proceduresinvolving, e.g., protein purification and protein visualization. Such anepitope-binding region is operably-linked in-frame to a modifiedClostridial toxin as a fusion protein. Non-limiting examples of anepitope-binding region include, e.g., FLAG, Express™ (SEQ ID NO: 108),human Influenza virus hemaglutinin (HA) (SEQ ID NO: 109), humanp62^(c-Myc) protein (c-MYC) (SEQ ID NO: 110), Vesicular Stomatitis VirusGlycoprotein (VSV-G) (SEQ ID NO: 111), Substance P (SEQ ID NO: 112),glycoprotein-D precursor of Herpes simplex virus (HSV) (SEQ ID NO: 113),V5 (SEQ ID NO: 114), AU1 (SEQ ID NO: 115) and AU5 (SEQ ID NO: 116);affinity-binding, such as, e.g., polyhistidine (HIS) (SEQ ID NO: 117),streptavidin binding peptide (strep), and biotin or a biotinylationsequence; peptide-binding regions, such as, e.g., the glutathionebinding domain of glutathione-S-transferase, the calmodulin bindingdomain of the calmodulin binding protein, and the maltose binding domainof the maltose binding protein. Non-limiting examples of specificprotocols for selecting, making and using an appropriate binding peptideare described in, e.g., Epitope Tagging, pp. 17.90-17.93 (Sambrook andRussell, eds., Molecular Cloning A Laboratory Manual, Vol. 3, 3^(rd) ed.2001); Antibodies: A Laboratory Manual (Edward Harlow & David Lane,eds., Cold Spring Harbor Laboratory Press, 2^(nd) ed. 1998); and UsingAntibodies: A Laboratory Manual: Portable Protocol No. I (Edward Harlow& David Lane, Cold Spring Harbor Laboratory Press, 1998). In addition,non-limiting examples of binding peptides as well as well-characterizedreagents, conditions and protocols are readily available from commercialvendors that include, without limitation, BD Biosciences-Clontech, PaloAlto, Calif.; BD Biosciences Pharmingen, San Diego, Calif.; Invitrogen,Inc, Carlsbad, Calif.; QIAGEN, Inc., Valencia, Calif.; and Stratagene,La Jolla, Calif. These protocols are routine procedures well within thescope of one skilled in the art and from the teaching herein.

Thus, in an embodiment, a modified Clostridial toxin disclosed in thepresent specification can further comprise an epitope-binding region. Inanother embodiment, a modified Clostridial toxin disclosed in thepresent specification can further comprises a plurality ofepitope-binding regions. In aspects of this embodiment, a modifiedClostridial toxin can comprise, e.g., at least 1 epitope-binding region,at least 2 epitope-binding regions, at least 3 epitope-binding regions,at least 4 epitope-binding regions or at least 5 epitope-bindingregions. In other aspects of this embodiment, a modified Clostridialtoxin can comprise, e.g., at most 1 epitope-binding region, at most 2epitope-binding regions, at most 3 epitope-binding regions, at most 4epitope-binding regions or at most 5 epitope-binding regions. In anotheraspect of this embodiment, a modified Clostridial toxin can comprise oneor more copies of the same epitope-binding region, one or more copies ofdifferent epitope-binding regions, or any combination thereof.

The location of an epitope-binding region can be in various positions,including, without limitation, at the amino terminus of a modifiedClostridial toxin, within a modified Clostridial toxin, or at thecarboxyl terminus of a modified Clostridial toxin. Thus, in anembodiment, an epitope-binding region is located at the amino-terminusof a modified Clostridial toxin. In such a location, a start methionineshould be placed in front of the epitope-binding region. In addition, itis known in the art that when adding a polypeptide that isoperationally-linked to the amino terminus of another polypeptidecomprising the start methionine that the original methionine residue canbe deleted. This is due to the fact that the added polypeptide willcontain a new start methionine and that the original start methioninemay reduce optimal expression of the fusion protein. In aspects of thisembodiment, an epitope-binding region located at the amino-terminus of amodified Clostridial toxin disclosed in the present specification canbe, e.g., a FLAG, Express™ epitope-binding region, a human Influenzavirus hemaglutinin (HA) epitope-binding region, a human p62^(c-Myc)protein (c-MYC) epitope-binding region, a Vesicular Stomatitis VirusGlycoprotein (VSV-G) epitope-binding region, a Substance Pepitope-binding region, a glycoprotein-D precursor of Herpes simplexvirus (HSV) epitope-binding region, a V5 epitope-binding region, a AU1epitope-binding region, a AU5 epitope-binding region, a polyhistidineepitope-binding region, a streptavidin binding peptide epitope-bindingregion, a biotin epitope-binding region, a biotinylation epitope-bindingregion, a glutathione binding domain of glutathione-S-transferase, acalmodulin binding domain of the calmodulin binding protein or a maltosebinding domain of the maltose binding protein.

In another embodiment, an epitope-binding region is located at thecarboxyl-terminus of a modified Clostridial toxin. In aspects of thisembodiment, an epitope-binding region located at the carboxyl-terminusof a modified Clostridial toxin disclosed in the present specificationcan be, e.g., a FLAG, Express™ epitope-binding region, a human Influenzavirus hemaglutinin (HA) epitope-binding region, a human p62^(c-Myc)protein (c-MYC) epitope-binding region, a Vesicular Stomatitis VirusGlycoprotein (VSV-G) epitope-binding region, a Substance Pepitope-binding region, a glycoprotein-D precursor of Herpes simplexvirus (HSV) epitope-binding region, a V5 epitope-binding region, a AU1epitope-binding region, a AU5 epitope-binding region, a polyhistidineepitope-binding region, a streptavidin binding peptide epitope-bindingregion, a biotin epitope-binding region, a biotinylation epitope-bindingregion, a glutathione binding domain of glutathione-S-transferase, acalmodulin binding domain of the calmodulin binding protein or a maltosebinding domain of the maltose binding protein.

It is envisioned that a modified Clostridial toxin disclosed in thepresent specification can comprise an enhance binding domain in any andall locations with the proviso that modified Clostridial toxin iscapable of performing the intoxication process. Non-limiting examplesinclude, locating an enhance binding domain at the amino terminus of amodified Clostridial toxin (FIG. 3); locating an enhance binding domainbetween a Clostridial toxin enzymatic domain and a Clostridial toxintranslocation domain of a modified Clostridial toxin (FIG. 4); andlocating an enhance binding domain at the carboxyl terminus of amodified Clostridial toxin (FIG. 5).

Thus, in an embodiment, a modified Clostridial toxin can comprise anamino to carboxyl single polypeptide linear order comprising aClostridial toxin enzymatic domain, a Clostridial toxin translocationdomain and an enhanced targeting domain. In an aspect of thisembodiment, a modified Clostridial toxin can comprise an amino tocarboxyl single polypeptide linear order comprising a Clostridial toxinenzymatic domain, a protease cleavage site, a Clostridial toxintranslocation domain and an enhanced targeting domain. In another aspectof this embodiment, a modified Clostridial toxin can comprise an aminoto carboxyl single polypeptide linear order comprising a Clostridialtoxin enzymatic domain, an endogenous protease cleavage site, aClostridial toxin translocation domain and an enhanced targeting domain.In another aspect of this embodiment, a modified Clostridial toxin cancomprise an amino to carboxyl single polypeptide linear order comprisinga Clostridial toxin enzymatic domain, an exogenous protease cleavagesite, a Clostridial toxin translocation domain and an enhanced targetingdomain.

In another embodiment, a modified Clostridial toxin can comprise anamino to carboxyl single polypeptide linear order comprising aClostridial toxin enzymatic domain, an enhanced targeting domain and aClostridial toxin translocation domain. In an aspect of this embodiment,a modified Clostridial toxin can comprise an amino to carboxyl singlepolypeptide linear order comprising a Clostridial toxin enzymaticdomain, a protease cleavage site, an enhanced targeting domain and aClostridial toxin translocation domain. In another aspect of thisembodiment, a modified Clostridial toxin can comprise an amino tocarboxyl single polypeptide linear order comprising a Clostridial toxinenzymatic domain, an endogenous protease cleavage site, an enhancedtargeting domain and a Clostridial toxin translocation domain. Inanother aspect of this embodiment, a modified Clostridial toxin cancomprise an amino to carboxyl single polypeptide linear order comprisinga Clostridial toxin enzymatic domain, an exogenous protease cleavagesite, an enhanced targeting domain and a Clostridial toxin translocationdomain.

In another embodiment, a modified Clostridial toxin can comprise anamino to carboxyl single polypeptide linear order comprising an enhancedtargeting domain, a Clostridial toxin translocation domain, and aClostridial toxin enzymatic domain. In an aspect of this embodiment, amodified Clostridial toxin can comprise an amino to carboxyl singlepolypeptide linear order comprising an enhanced targeting domain, aClostridial toxin translocation domain, a protease cleavage site and aClostridial toxin enzymatic domain. In another aspect of thisembodiment, a modified Clostridial toxin can comprise an amino tocarboxyl single polypeptide linear order comprising an enhancedtargeting domain, a Clostridial toxin translocation domain, anendogenous protease cleavage site and a Clostridial toxin enzymaticdomain. In another aspect of this embodiment, a modified Clostridialtoxin can comprise an amino to carboxyl single polypeptide linear ordercomprising an enhanced targeting domain, a Clostridial toxintranslocation domain, an exogenous protease cleavage site and aClostridial toxin enzymatic domain.

In another embodiment, a modified Clostridial toxin can comprise anamino to carboxyl single polypeptide linear order comprising an enhancedtargeting domain, a Clostridial toxin enzymatic domain and a Clostridialtoxin translocation domain. In an aspect of this embodiment, a modifiedClostridial toxin can comprise an amino to carboxyl single polypeptidelinear order comprising an enhanced targeting domain, a Clostridialtoxin enzymatic domain, a protease cleavage site and a Clostridial toxintranslocation domain. In another aspect of this embodiment, a modifiedClostridial toxin can comprise an amino to carboxyl single polypeptidelinear order comprising an enhanced targeting domain, a Clostridialtoxin enzymatic domain, an endogenous protease cleavage site and aClostridial toxin translocation domain. In another aspect of thisembodiment, a modified Clostridial toxin can comprise an amino tocarboxyl single polypeptide linear order comprising an enhancedtargeting domain, a Clostridial toxin enzymatic domain, an exogenousprotease cleavage site and a Clostridial toxin translocation domain.

In another embodiment, a modified Clostridial toxin can comprise anamino to carboxyl single polypeptide linear order comprising aClostridial toxin translocation domain, a Clostridial toxin enzymaticdomain and an enhanced targeting domain. In an aspect of thisembodiment, a modified Clostridial toxin can comprise an amino tocarboxyl single polypeptide linear order comprising a Clostridial toxintranslocation domain, a protease cleavage site, a Clostridial toxinenzymatic domain and an enhanced targeting domain. In another aspect ofthis embodiment, a modified Clostridial toxin can comprise an amino tocarboxyl single polypeptide linear order comprising a Clostridial toxintranslocation domain, an endogenous protease cleavage site, aClostridial toxin enzymatic domain and an enhanced targeting domain. Inanother aspect of this embodiment, a modified Clostridial toxin cancomprise an amino to carboxyl single polypeptide linear order comprisinga Clostridial toxin translocation domain, an exogenous protease cleavagesite, a Clostridial toxin enzymatic domain and an enhanced targetingdomain.

Aspects of the present invention provide, in part modified Clostridialtoxins. Non-limiting examples of Clostridial toxin modificationsdisclosed in the present specification include, e.g., addition of anenhanced targeting domain, addition of a protease cleavage site,rearrangement of the enzymatic, translocation and binding domains andaddition of a spacer region. It is understood that all suchmodifications do not substantially affect the ability of a modifiedClostridial toxin to intoxicate a cell. As used herein, the term “do notsubstantially affect” means a modified Clostridial toxin can stillexecute the overall cellular mechanism whereby a Clostridial toxinenters a neuron and inhibits neurotransmitter release and encompassesthe binding of a Clostridial toxin to a low or high affinity receptorcomplex, the internalization of the toxin/receptor complex, thetranslocation of the Clostridial toxin light chain into the cytoplasmand the enzymatic modification of a Clostridial toxin substrate. Inaspects of this embodiment, the modified Clostridial toxin is, e.g., atleast 10% as toxic as a naturally-occurring Clostridial toxin, at least20% as toxic as a naturally-occurring Clostridial toxin, at least 30% astoxic as a naturally-occurring Clostridial toxin, at least 40% as toxicas a naturally-occurring Clostridial toxin, at least 50% as toxic as anaturally-occurring Clostridial toxin, at least 60% as toxic as anaturally-occurring Clostridial toxin, at least 70% as toxic as anaturally-occurring Clostridial toxin, at least 80% as toxic as anaturally-occurring Clostridial toxin, at least 90% as toxic as anaturally-occurring Clostridial toxin or at least 95% as toxic as anaturally-occurring Clostridial toxin. In aspects of this embodiment,the modified Clostridial toxin is, e.g., at most 10% as toxic as anaturally-occurring Clostridial toxin, at most 20% as toxic as anaturally-occurring Clostridial toxin, at most 30% as toxic as anaturally-occurring Clostridial toxin, at most 40% as toxic as anaturally-occurring Clostridial toxin, at most 50% as toxic as anaturally-occurring Clostridial toxin, at most 60% as toxic as anaturally-occurring Clostridial toxin, at most 70% as toxic as anaturally-occurring Clostridial toxin, at most 80% as toxic as anaturally-occurring Clostridial toxin, at most 90% as toxic as anaturally-occurring Clostridial toxin or at most 95% as toxic as anaturally-occurring Clostridial toxin.

Another aspect of the present invention provides polynucleotidemolecules encoding modified Clostridial toxins disclosed in the presentspecification. It is envisioned that any and all modified Clostridialtoxin disclosed in the present specification can be encoded by apolynucleotide molecule.

Aspects of the present invention provide, in part polynucleotidemolecules. As used herein, the term “polynucleotide molecule” issynonymous with “nucleic acid molecule” and means a polymeric form ofnucleotides, such as, e.g., ribonucleotides and deoxyribonucleotides, ofany length. It is envisioned that any and all polynucleotide moleculesthat can encode a modified Clostridial toxin disclosed in the presentspecification can be useful, including, without limitationnaturally-occurring and non-naturally-occurring DNA molecules andnaturally-occurring and non-naturally-occurring RNA molecules.Non-limiting examples of naturally-occurring and non-naturally-occurringDNA molecules include single-stranded DNA molecules, double-stranded DNAmolecules, genomic DNA molecules, cDNA molecules, vector constructs,such as, e.g., plasmid constructs, phagmid constructs, bacteriophageconstructs, retroviral constructs and artificial chromosome constructs.Non-limiting examples of naturally-occurring and non-naturally-occurringRNA molecules include single-stranded RNA, double stranded RNA and mRNA.

Thus, in an embodiment, a polynucleotide molecule encodes a modifiedClostridial toxin disclosed in the present specification.

In another embodiment, a polynucleotide molecule encodes, in part, amodified Clostridial toxin comprising a Clostridial toxin enzymaticdomain disclosed in the present specification. In an aspect of thisembodiment, a polynucleotide molecule encoding a modified Clostridialtoxin enzymatic domain comprises a naturally occurring Clostridial toxinenzymatic domain variant, such as, e.g., a Clostridial toxin enzymaticdomain isoform or a Clostridial toxin enzymatic domain subtype. Inanother aspect of this embodiment, a polynucleotide molecule encoding aClostridial toxin enzymatic domain comprises a non-naturally occurringClostridial toxin enzymatic domain variant, such as, e.g., aconservative Clostridial toxin enzymatic domain variant, anon-conservative Clostridial toxin enzymatic domain variant or an activeClostridial toxin enzymatic domain fragment, or any combination thereof.In other aspects of this embodiment, a polynucleotide molecule encodinga Clostridial toxin enzymatic domain comprises a BoNT/A enzymaticdomain, a BoNT/B enzymatic domain, a BoNT/C1 enzymatic domain, a BoNT/Denzymatic domain, a BoNT/E enzymatic domain, a BoNT/F enzymatic domain,a BoNT/G enzymatic domain, a TeNT enzymatic domain, or active fragmentthereof.

In another embodiment, a polynucleotide molecule encodes, in part, amodified Clostridial toxin comprising a Clostridial toxin translocationdomain disclosed in the present specification. In an aspect of thisembodiment, a polynucleotide molecule encoding a modified Clostridialtoxin translocation domain comprises a naturally occurring Clostridialtoxin translocation domain variant, such as, e.g., a Clostridial toxintranslocation domain isoform or a Clostridial toxin translocation domainsubtype. In another aspect of this embodiment, a polynucleotide moleculeencoding a Clostridial toxin translocation domain comprises anon-naturally occurring Clostridial toxin translocation domain variant,such as, e.g., a conservative Clostridial toxin translocation domainvariant, a non-conservative Clostridial toxin translocation domainvariant or an active Clostridial toxin translocation domain fragment, orany combination thereof. In other aspects of this embodiment, apolynucleotide molecule encoding a Clostridial toxin translocationdomain comprises a BoNT/A translocation domain, a BoNT/B translocationdomain, a BoNT/C1 translocation domain, a BoNT/D translocation domain, aBoNT/E translocation domain, a BoNT/F translocation domain, a BoNT/Gtranslocation domain, a TeNT translocation domain, or active fragmentthereof.

In another embodiment, a polynucleotide molecule encodes, in part, amodified Clostridial toxin comprising an enhanced targeting domaindisclosed in the present specification. In an aspect of this embodiment,a polynucleotide molecule encoding an enhanced targeting domaincomprises a polypeptide that selectively binds to a non-Clostridialtoxin receptor system present on a presynaptic membrane of a Clostridialtoxin target cell. In an aspect of this embodiment, a polynucleotidemolecule encoding a polypeptide that selectively binds to anon-Clostridial toxin receptor system present on a presynaptic membraneof a Clostridial toxin target cell comprises a naturally occurringvariant, such as, e.g., an isoform or a subtype. In another aspect ofthis embodiment, a polynucleotide molecule encoding a polypeptide thatselectively binds to a non-Clostridial toxin receptor system present ona presynaptic membrane of a Clostridial toxin target cell comprises anon-naturally occurring variant, such as, e.g., a conservative variant,a non-conservative variant or an active fragment, or any combinationthereof. In other aspects of this embodiment, a polynucleotide moleculeencoding a polypeptide that selectively binds to a non-Clostridial toxinreceptor system present on a presynaptic membrane of a Clostridial toxintarget cell comprises a TGFβ superfamily polypeptide, such as, e.g., aTGFβ, a BMPs, a GDF and an activin; a neurotrophin, such as, e.g., aNGF, a BDNF, a NT-3 and a NT-4/5; an axon guidance signaling molecule,such as, e.g., a netrin, a semaphoring and an ephrin; a neuroregulatorycytokine, such as, e.g., a CNTF, a GPA, a LIF, an interleukin, anonostatin M, a CT-1 and a CLC; a sugar binding protein, such as, e.g., aserum amyloid P, a β-glucanase, a sialidase, a lectin, a cryia, aninsecticidal delta-endotoxin, an agglutinin, an abrin and a ricin; anIGF, such as, e.g., a IGF-1 and a IGF-2; a neurexin; aneuroleukin/AMF/MF; a TrkB; an EGF; a visceral gut peptide such as,e.g., a gastrin, a VIP, a bombesin; and a WNT, such as, e.g., aFrizzled.

In another embodiment, a polynucleotide molecule encodes, in part, amodified Clostridial toxin comprising an enhanced targeting domaindisclosed in the present specification. In an aspect of this embodiment,a polynucleotide molecule encoding an enhanced targeting domaincomprises a polypeptide that selectively binds to a non-Clostridialtoxin receptor system present on a postsynaptic membrane of aClostridial toxin target cell. In an aspect of this embodiment, apolynucleotide molecule encoding a polypeptide that selectively binds toa non-Clostridial toxin receptor system present on a postsynapticmembrane of a Clostridial toxin target cell comprises a naturallyoccurring variant, such as, e.g., an isoform or a subtype. In anotheraspect of this embodiment, a polynucleotide molecule encoding apolypeptide that selectively binds to a non-Clostridial toxin receptorsystem present on a postsynaptic membrane of a Clostridial toxin targetcell comprises a non-naturally occurring variant, such as, e.g., aconservative variant, a non-conservative variant or an active fragment,or any combination thereof. In other aspects of this embodiment, apolynucleotide molecule encoding a polypeptide that selectively binds toa non-Clostridial toxin receptor system present on a postsynapticmembrane of a Clostridial toxin target cell comprises a Ng-CAM(L1), aN-CAM, a N-cadherin, an agrin-MUSK, and a basement membrane polypeptide,such as, e.g., laminin β-2.

In another embodiment, a polynucleotide molecule encodes, in part, amodified Clostridial toxin comprising a protease cleavage site disclosedin the present specification. In an aspect of this embodiment, apolynucleotide molecule encoding a protease cleavage site comprises anendogenous Clostridial toxin protease site. In aspects of thisembodiment, a polynucleotide molecule encoding an endogenous Clostridialtoxin protease site can be, e.g., a BoNT/A di-chain loop proteasecleavage site, a BoNT/B di-chain loop protease cleavage site, a BoNT/C1di-chain loop protease cleavage site, a BoNT/D di-chain loop proteasecleavage site, a BoNT/E di-chain loop protease cleavage site, a BoNT/Fdi-chain loop protease cleavage site, a BoNT/G di-chain loop proteasecleavage site or a TeNT di-chain loop protease cleavage site. In anotheraspect of this embodiment, a polynucleotide molecule encoding a proteasecleavage site comprises an exogenous Clostridial toxin protease site. Inaspects of this embodiment, a polynucleotide molecule encoding anexogenous Clostridial toxin protease site can be, e.g., a bovineenterokinase protease cleavage site, a Tobacco Etch Virus proteasecleavage site, a Human Rhinovirus 3C protease cleavage site, aSUMO/ULP-1 protease cleavage site, a Thrombin protease cleavage site, aCoagulation Factor Xa protease cleavage site or a Clostridial toxinsubstrate cleavage site, such as, e.g., a BoNT/A substrate cleavagesite, a BoNT/B substrate cleavage site, a BoNT/C1 substrate cleavagesite, a BoNT/D substrate cleavage site, a BoNT/E substrate cleavagesite, a BoNT/F substrate cleavage site, a BoNT/G substrate cleavage siteor a TeNT substrate cleavage site.

In another embodiment, a polynucleotide molecule encodes, in part, amodified Clostridial toxin comprising a flexible spacer disclosed in thepresent specification. In an aspect of this embodiment, a polynucleotidemolecule encoding a flexible spacer a G-spacer, a A-spacer of anycombination thereof.

Well-established molecular biology techniques that may be necessary tomake a polynucleotide molecule encoding a modified Clostridial toxindisclosed in the present specification including, but not limited to,procedures involving polymerase chain reaction (PCR) amplification,restriction enzyme reactions, agarose gel electrophoresis, nucleic acidligation, bacterial transformation, nucleic acid purification, nucleicacid sequencing and recombination-based techniques are routineprocedures well within the scope of one skilled in the art and from theteaching herein. Non-limiting examples of specific protocols necessaryto make a polynucleotide molecule encoding a modified Clostridial toxinare described in e.g., MOLECULAR CLONING A LABORATORY MANUAL, supra,(2001); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Frederick M. Ausubelet al., eds. John Wiley & Sons, 2004). Additionally, a variety ofcommercially available products useful for making a polynucleotidemolecule encoding a modified Clostridial toxin are widely available.These protocols are routine procedures well within the scope of oneskilled in the art and from the teaching herein.

Another aspect of the present invention provides a method of producing amodified Clostridial toxin disclosed in the present specification, suchmethod comprising the step of expressing a polynucleotide moleculeencoding a modified Clostridial toxin in a cell. Another aspect of thepresent invention provides a method of producing a modified Clostridialtoxin disclosed in the present specification, such method comprising thesteps of introducing an expression construct comprising a polynucleotidemolecule encoding a modified Clostridial toxin into a cell andexpressing the expression construct in the cell.

The methods disclosed in the present specification include, in part, amodified Clostridial toxin. It is envisioned that any and all modifiedClostridial toxins disclosed in the present specification can beproduced using the methods disclosed in the present specification. It isalso envisioned that any and all polynucleotide molecules encoding amodified Clostridial toxins disclosed in the present specification canbe useful in producing a modified Clostridial toxins disclosed in thepresent specification using the methods disclosed in the presentspecification.

The methods disclosed in the present specification include, in part, anexpression construct. An expression construct comprises a polynucleotidemolecule disclosed in the present specification operably-linked to anexpression vector useful for expressing the polynucleotide molecule in acell or cell-free extract. A wide variety of expression vectors can beemployed for expressing a polynucleotide molecule encoding a modifiedClostridial toxin, including, without limitation, a viral expressionvector; a prokaryotic expression vector; eukaryotic expression vectors,such as, e.g., a yeast expression vector, an insect expression vectorand a mammalian expression vector; and a cell-free extract expressionvector. It is further understood that expression vectors useful topractice aspects of these methods may include those which express amodified Clostridial toxin under control of a constitutive,tissue-specific, cell-specific or inducible promoter element, enhancerelement or both. Non-limiting examples of expression vectors, along withwell-established reagents and conditions for making and using anexpression construct from such expression vectors are readily availablefrom commercial vendors that include, without limitation, BDBiosciences-Clontech, Palo Alto, Calif.; BD Biosciences Pharmingen, SanDiego, Calif.; Invitrogen, Inc, Carlsbad, Calif.; EMDBiosciences-Novagen, Madison, Wis.; QIAGEN, Inc., Valencia, Calif.; andStratagene, La Jolla, Calif. The selection, making and use of anappropriate expression vector are routine procedures well within thescope of one skilled in the art and from the teachings herein.

Thus, aspects of this embodiment include, without limitation, a viralexpression vector operably-linked to a polynucleotide molecule encodinga modified Clostridial toxin; a prokaryotic expression vectoroperably-linked to a polynucleotide molecule encoding a modifiedClostridial toxin; a yeast expression vector operably-linked to apolynucleotide molecule encoding a modified Clostridial toxin; an insectexpression vector operably-linked to a polynucleotide molecule encodinga modified Clostridial toxin; and a mammalian expression vectoroperably-linked to a polynucleotide molecule encoding a modifiedClostridial toxin. Other aspects of this embodiment include, withoutlimitation, expression constructs suitable for expressing a modifiedClostridial toxin disclosed in the present specification using acell-free extract comprising a cell-free extract expression vectoroperably linked to a polynucleotide molecule encoding a modifiedClostridial toxin.

The methods disclosed in the present specification include, in part, acell. It is envisioned that any and all cells can be used. Thus, aspectsof this embodiment include, without limitation, prokaryotic cellsincluding, without limitation, strains of aerobic, microaerophilic,capnophilic, facultative, anaerobic, gram-negative and gram-positivebacterial cells such as those derived from, e.g., Escherichia coli,Bacillus subtilis, Bacillus licheniformis, Bacteroides fragilis,Clostridia perfringens, Clostridia difficile, Caulobacter crescentus,Lactococcus lactis, Methylobacterium extorquens, Neisseria meningirulls,Neisseria meningitidis, Pseudomonas fluorescens and Salmonellatyphimurium; and eukaryotic cells including, without limitation, yeaststrains, such as, e.g., those derived from Pichia pastoris, Pichiamethanolica, Pichia angusta, Schizosaccharomyces pombe, Saccharomycescerevisiae and Yarrowia lipolytica; insect cells and cell lines derivedfrom insects, such as, e.g., those derived from Spodoptera frugiperda,Trichoplusia ni, Drosophila melanogaster and Manduca sexta; andmammalian cells and cell lines derived from mammalian cells, such as,e.g., those derived from mouse, rat, hamster, porcine, bovine, equine,primate and human. Cell lines may be obtained from the American TypeCulture Collection, European Collection of Cell Cultures and the GermanCollection of Microorganisms and Cell Cultures. Non-limiting examples ofspecific protocols for selecting, making and using an appropriate cellline are described in e.g., INSECT CELL CULTURE ENGINEERING (Mattheus F.A. Goosen et al. eds., Marcel Dekker, 1993); INSECT CELL CULTURES:FUNDAMENTAL AND APPLIED ASPECTS (J. M. Vlak et al. eds., Kluwer AcademicPublishers, 1996); Maureen A. Harrison & Ian F. Rae, GENERAL TECHNIQUESOF CELL CULTURE (Cambridge University Press, 1997); CELL AND TISSUECULTURE: LABORATORY PROCEDURES (Alan Doyle et al eds., John Wiley andSons, 1998); R. Ian Freshney, CULTURE OF ANIMAL CELLS: A MANUAL OF BASICTECHNIQUE (Wiley-Liss, 4^(th) ed. 2000); ANIMAL CELL CULTURE: APRACTICAL APPROACH (John R. W. Masters ed., Oxford University Press,3^(rd) ed. 2000); MOLECULAR CLONING A LABORATORY MANUAL, supra, (2001);BASIC CELL CULTURE: A PRACTICAL APPROACH (John M. Davis, Oxford Press,2^(nd) ed. 2002); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, supra,(2004). These protocols are routine procedures within the scope of oneskilled in the art and from the teaching herein.

The methods disclosed in the present specification include, in part,introducing into a cell a polynucleotide molecule. A polynucleotidemolecule introduced into a cell can be transiently or stably maintainedby that cell. Stably-maintained polynucleotide molecules may beextra-chromosomal and replicate autonomously, or they may be integratedinto the chromosomal material of the cell and replicatenon-autonomously. It is envisioned that any and all methods forintroducing a polynucleotide molecule disclosed in the presentspecification into a cell can be used. Methods useful for introducing anucleic acid molecule into a cell include, without limitation,chemical-mediated transfection such as, e.g., calciumphosphate-mediated, diethyl-aminoethyl (DEAE) dextran-mediated,lipid-mediated, polyethyleneimine (PEI)-mediated, polylysine-mediatedand polybrene-mediated; physical-mediated transfection, such as, e.g.,biolistic particle delivery, microinjection, protoplast fusion andelectroporation; and viral-mediated transfection, such as, e.g.,retroviral-mediated transfection, see, e.g., Introducing Cloned Genesinto Cultured Mammalian Cells, pp. 16.1-16.62 (Sambrook & Russell, eds.,Molecular Cloning A Laboratory Manual, Vol. 3, 3^(rd) ed. 2001). Oneskilled in the art understands that selection of a specific method tointroduce an expression construct into a cell will depend, in part, onwhether the cell will transiently contain an expression construct orwhether the cell will stably contain an expression construct. Theseprotocols are routine procedures within the scope of one skilled in theart and from the teaching herein.

In an aspect of this embodiment, a chemical-mediated method, termedtransfection, is used to introduce a polynucleotide molecule encoding amodified Clostridial toxin into a cell. In chemical-mediated methods oftransfection the chemical reagent forms a complex with the nucleic acidthat facilitates its uptake into the cells. Such chemical reagentsinclude, without limitation, calcium phosphate-mediated, see, e.g.,Martin Jordan & Florian Worm, Transfection of adherent and suspendedcells by calcium phosphate, 33(2) Methods 136-143 (2004);diethyl-aminoethyl (DEAE) dextran-mediated, lipid-mediated, cationicpolymer-mediated like polyethyleneimine (PEI)-mediated andpolylysine-mediated and polybrene-mediated, see, e.g., Chun Zhang etal., Polyethylenimine strategies for plasmid delivery to brain-derivedcells, 33(2) Methods 144-150 (2004). Such chemical-mediated deliverysystems can be prepared by standard methods and are commerciallyavailable, see, e.g., CellPhect Transfection Kit (Amersham Biosciences,Piscataway, N.J.); Mammalian Transfection Kit, Calcium phosphate andDEAE Dextran, (Stratagene, Inc., La Jolla, Calif.); Lipofectamine™Transfection Reagent (Invitrogen, Inc., Carlsbad, Calif.); ExGen 500Transfection kit (Fermentas, Inc., Hanover, Md.), and SuperFect andEffectene Transfection Kits (Qiagen, Inc., Valencia, Calif.).

In another aspect of this embodiment, a physical-mediated method is usedto introduce a polynucleotide molecule encoding a modified Clostridialtoxin into a cell. Physical techniques include, without limitation,electroporation, biolistic and microinjection. Biolistics andmicroinjection techniques perforate the cell wall in order to introducethe nucleic acid molecule into the cell, see, e.g., Jeike E. Biewenga etal., Plasmid-mediated gene transfer in neurons using the biolisticstechnique, 71(1) J. Neurosci. Methods. 67-75 (1997); and John O'Brien &Sarah C. R. Lummis, Biolistic and diolistic transfection: using the genegun to deliver DNA and lipophilic dyes into mammalian cells, 33(2)Methods 121-125 (2004). Electroporation, also termedelectropermeabilization, uses brief, high-voltage, electrical pulses tocreate transient pores in the membrane through which the nucleic acidmolecules enter and can be used effectively for stable and transienttransfections of all cell types, see, e.g., M. Golzio et al., In vitroand in vivo electric field-mediated permeabilization, gene transfer, andexpression, 33(2) Methods 126-135 (2004); and Oliver Gresch et al., Newnon-viral method for gene transfer into primary cells, 33(2) Methods151-163 (2004).

In another aspect of this embodiment, a viral-mediated method, termedtransduction, is used to introduce a polynucleotide molecule encoding amodified Clostridial toxin into a cell. In viral-mediated methods oftransient transduction, the process by which viral particles infect andreplicate in a host cell has been manipulated in order to use thismechanism to introduce a nucleic acid molecule into the cell.Viral-mediated methods have been developed from a wide variety ofviruses including, without limitation, retroviruses, adenoviruses,adeno-associated viruses, herpes simplex viruses, picornaviruses,alphaviruses and baculoviruses, see, e.g., Armin Blesch, Lentiviral andMLV based retroviral vectors for ex vivo and in vivo gene transfer,33(2) Methods 164-172 (2004); and Maurizio Federico, From lentivirusesto lentivirus vectors, 229 Methods Mol. Biol. 3-15 (2003); E. M.Poeschla, Non-primate lentiviral vectors, 5(5) Curr. Opin. Mol. Ther.529-540 (2003); Karim Benihoud et al, Adenovirus vectors for genedelivery, 10(5) Curr. Opin. Biotechnol. 440-447 (1999); H. Bueler,Adeno-associated viral vectors for gene transfer and gene therapy,380(6) Biol. Chem. 613-622 (1999); Chooi M. Lai et al., Adenovirus andadeno-associated virus vectors, 21(12) DNA Cell Biol. 895-913 (2002);Edward A. Burton et al., Gene delivery using herpes simplex virusvectors, 21(12) DNA Cell Biol. 915-936 (2002); Paola Grandi et al.,Targeting HSV amplicon vectors, 33(2) Methods 179-186 (2004); IlyaFrolov et al., Alphavirus-based expression vectors: strategies andapplications, 93(21) Proc. Natl. Acad. Sci. U.S.A. 11371-11377 (1996);Markus U. Ehrengruber, Alphaviral gene transfer in neurobiology, 59(1)Brain Res. Bull. 13-22 (2002); Thomas A. Kost & J. Patrick Condreay,Recombinant baculoviruses as mammalian cell gene-delivery vectors, 20(4)Trends Biotechnol. 173-180 (2002); and A. Huser & C. Hofmann,Baculovirus vectors: novel mammalian cell gene-delivery vehicles andtheir applications, 3(1) Am. J. Pharmacogenomics 53-63 (2003).

Adenoviruses, which are non-enveloped, double-stranded DNA viruses, areoften selected for mammalian cell transduction because adenoviruseshandle relatively large polynucleotide molecules of about 36 kb, areproduced at high titer, and can efficiently infect a wide variety ofboth dividing and non-dividing cells, see, e.g., Wim T. J. M. C. Hermenset al., Transient gene transfer to neurons and glia: analysis ofadenoviral vector performance in the CNS and PNS, 71(1) J. Neurosci.Methods 85-98 (1997); and Hiroyuki Mizuguchi et al., Approaches forgenerating recombinant adenovirus vectors, 52(3) Adv. Drug Deliv. Rev.165-176 (2001). Transduction using adenoviral-based system do notsupport prolonged protein expression because the nucleic acid moleculeis carried from an episome in the cell nucleus, rather than beingintegrated into the host cell chromosome. Adenoviral vector systems andspecific protocols for how to use such vectors are disclosed in, e.g.,ViraPower™ Adenoviral Expression System (Invitrogen, Inc., Carlsbad,Calif.) and ViraPower™ Adenoviral Expression System Instruction Manual25-0543 version A, Invitrogen, Inc., (Jul. 15, 2002); and AdEasy™Adenoviral Vector System (Stratagene, Inc., La Jolla, Calif.) andAdEasy™ Adenoviral Vector System Instruction Manual 064004f, Stratagene,Inc.

Nucleic acid molecule delivery can also use single-stranded RNAretroviruses, such as, e.g., oncoretroviruses and lentiviruses.Retroviral-mediated transduction often produce transduction efficienciesclose to 100%, can easily control the proviral copy number by varyingthe multiplicity of infection (MOI), and can be used to eithertransiently or stably transduce cells, see, e.g., Tiziana Tonini et al.,Transient production of retroviral- and lentiviral-based vectors for thetransduction of Mammalian cells, 285 Methods Mol. Biol. 141-148 (2004);Armin Blesch, Lentiviral and MLV based retroviral vectors for ex vivoand in vivo gene transfer, 33(2) Methods 164-172 (2004); FelixRecillas-Targa, Gene transfer and expression in mammalian cell lines andtransgenic animals, 267 Methods Mol. Biol. 417-433 (2004); and RolandWolkowicz et al., Lentiviral vectors for the delivery of DNA intomammalian cells, 246 Methods Mol. Biol. 391-411 (2004). Retroviralparticles consist of an RNA genome packaged in a protein capsid,surrounded by a lipid envelope. The retrovirus infects a host cell byinjecting its RNA into the cytoplasm along with the reversetranscriptase enzyme. The RNA template is then reverse transcribed intoa linear, double stranded cDNA that replicates itself by integratinginto the host cell genome. Viral particles are spread both vertically(from parent cell to daughter cells via the provirus) as well ashorizontally (from cell to cell via virions). This replication strategyenables long-term persistent expression since the nucleic acid moleculesof interest are stably integrated into a chromosome of the host cell,thereby enabling long-term expression of the protein. For instance,animal studies have shown that lentiviral vectors injected into avariety of tissues produced sustained protein expression for more than 1year, see, e.g., Luigi Naldini et al., In vivo gene delivery and stabletransduction of non-dividing cells by a lentiviral vector, 272(5259)Science 263-267 (1996). The Oncoretroviruses-derived vector systems,such as, e.g., Moloney murine leukemia virus (MoMLV), are widely usedand infect many different non-dividing cells. Lentiviruses can alsoinfect many different cell types, including dividing and non-dividingcells and possess complex envelope proteins, which allows for highlyspecific cellular targeting.

Retroviral vectors and specific protocols for how to use such vectorsare disclosed in, e.g., U.S. Patent Nos. Manfred Gossen & HermannBujard, Tight control of gene expression in eukaryotic cells bytetracycline-responsive promoters, U.S. Pat. No. 5,464,758 (Nov. 7,1995) and Hermann Bujard & Manfred Gossen, Methods for regulating geneexpression, U.S. Pat. No. 5,814,618 (Sep. 29, 1998) David S. Hogness,Polynucleotides encoding insect steroid hormone receptor polypeptidesand cells transformed with same, U.S. Pat. No. 5,514,578 (May 7, 1996)and David S. Hogness, Polynucleotide encoding insect ecdysone receptor,U.S. Pat. No. 6,245,531 (Jun. 12, 2001); Elisabetta Vegeto et al.,Progesterone receptor having C. terminal hormone binding domaintruncations, U.S. Pat. No. 5,364,791 (Nov. 15, 1994), Elisabetta Vegetoet al., Mutated steroid hormone receptors, methods for their use andmolecular switch for gene therapy, U.S. Pat. No. 5,874,534 (Feb. 23,1999) and Elisabetta Vegeto et al., Mutated steroid hormone receptors,methods for their use and molecular switch for gene therapy, U.S. Pat.No. 5,935,934 (Aug. 10, 1999). Furthermore, such viral delivery systemscan be prepared by standard methods and are commercially available, see,e.g., BD™ Tet-Off and Tet-On Gene Expression Systems (BDBiosciences-Clonetech, Palo Alto, Calif.) and BD™ Tet-Off and Tet-OnGene Expression Systems User Manual, PT3001-1, BD Biosciences Clonetech,(Mar. 14, 2003), GeneSwitch™ System (Invitrogen, Inc., Carlsbad, Calif.)and GeneSwitch™ System A Mifepristone-Regulated Expression System forMammalian Cells version D, 25-0313, Invitrogen, Inc., (Nov. 4, 2002);ViraPower™ Lentiviral Expression System (Invitrogen, Inc., Carlsbad,Calif.) and ViraPower™ Lentiviral Expression System Instruction Manual25-0501 version E, Invitrogen, Inc., (Dec. 8, 2003); and CompleteControl® Retroviral Inducible Mammalian Expression System (Stratagene,La Jolla, Calif.) and Complete Control® Retroviral Inducible MammalianExpression System Instruction Manual, 064005e.

The methods disclosed in the present specification include, in part,expressing a modified Clostridial toxin from a polynucleotide molecule.It is envisioned that any of a variety of expression systems may beuseful for expressing a modified Clostridial toxin from a polynucleotidemolecule disclosed in the present specification, including, withoutlimitation, cell-based systems and cell-free expression systems.Cell-based systems include, without limitation, viral expressionsystems, prokaryotic expression systems, yeast expression systems,baculoviral expression systems, insect expression systems and mammalianexpression systems. Cell-free systems include, without limitation, wheatgerm extracts, rabbit reticulocyte extracts and E. coli extracts andgenerally are equivalent to the method disclosed herein. Expression of apolynucleotide molecule using an expression system can include any of avariety of characteristics including, without limitation, inducibleexpression, non-inducible expression, constitutive expression,viral-mediated expression, stably-integrated expression, and transientexpression. Expression systems that include well-characterized vectors,reagents, conditions and cells are well-established and are readilyavailable from commercial vendors that include, without limitation,Ambion, Inc. Austin, Tex.; BD Biosciences-Clontech, Palo Alto, Calif.;BD Biosciences Pharmingen, San Diego, Calif.; Invitrogen, Inc, Carlsbad,Calif.; QIAGEN, Inc., Valencia, Calif.; Roche Applied Science,Indianapolis, Ind.; and Stratagene, La Jolla, Calif. Non-limitingexamples on the selection and use of appropriate heterologous expressionsystems are described in e.g., PROTEIN EXPRESSION. A PRACTICAL APPROACH(S. J. Higgins and B. David Hames eds., Oxford University Press, 1999);Joseph M. Fernandez & James P. Hoeffler, GENE EXPRESSION SYSTEMS. USINGNATURE FOR THE ART OF EXPRESSION (Academic Press, 1999); and Meena Rai &Harish Padh, Expression Systems for Production of Heterologous Proteins,80(9) CURRENT SCIENCE 1121-1128, (2001). These protocols are routineprocedures well within the scope of one skilled in the art and from theteaching herein.

A variety of cell-based expression procedures are useful for expressinga modified Clostridial toxin encoded by polynucleotide moleculedisclosed in the present specification. Examples included, withoutlimitation, viral expression systems, prokaryotic expression systems,yeast expression systems, baculoviral expression systems, insectexpression systems and mammalian expression systems. Viral expressionsystems include, without limitation, the ViraPower™ Lentiviral(Invitrogen, Inc., Carlsbad, Calif.), the Adenoviral Expression Systems(Invitrogen, Inc., Carlsbad, Calif.), the AdEasy™ XL Adenoviral VectorSystem (Stratagene, La Jolla, Calif.) and the ViraPort® Retroviral GeneExpression System (Stratagene, La Jolla, Calif.). Non-limiting examplesof prokaryotic expression systems include the Champion™ pET ExpressionSystem (EMD Biosciences-Novagen, Madison, Wis.), the TriEx™ BacterialExpression System (EMD Biosciences-Novagen, Madison, Wis.), theQIAexpress® Expression System (QIAGEN, Inc.), and the Affinity® ProteinExpression and Purification System (Stratagene, La Jolla, Calif.). Yeastexpression systems include, without limitation, the EasySelect™ PichiaExpression Kit (Invitrogen, Inc., Carlsbad, Calif.), the YES-Echo™Expression Vector Kits (Invitrogen, Inc., Carlsbad, Calif.) and theSpECTRA™ S. pombe Expression System (Invitrogen, Inc., Carlsbad,Calif.). Non-limiting examples of baculoviral expression systems includethe BaculoDirect™ (Invitrogen, Inc., Carlsbad, Calif.), the Bac-to-Bac®(Invitrogen, Inc., Carlsbad, Calif.), and the BD BaculoGold™ (BDBiosciences-Pharmigen, San Diego, Calif.). Insect expression systemsinclude, without limitation, the Drosophila Expression System (DES®)(Invitrogen, Inc., Carlsbad, Calif.), InsectSelect™ System (Invitrogen,Inc., Carlsbad, Calif.) and InsectDirect™ System (EMDBiosciences-Novagen, Madison, Wis.). Non-limiting examples of mammalianexpression systems include the T-Rex™ (Tetracycline-RegulatedExpression) System (Invitrogen, Inc., Carlsbad, Calif.), the Flp-In™T-REx™ System (Invitrogen, Inc., Carlsbad, Calif.), the pcDNA™ system(Invitrogen, Inc., Carlsbad, Calif.), the pSecTag2 system (Invitrogen,Inc., Carlsbad, Calif.), the Exchanger® System, InterPlay™ Mammalian TAPSystem (Stratagene, La Jolla, Calif.), Complete Control® InducibleMammalian Expression System (Stratagene, La Jolla, Calif.) andLacSwitch® II Inducible Mammalian Expression System (Stratagene, LaJolla, Calif.).

Another procedure of expressing a modified Clostridial toxin encoded bypolynucleotide molecule disclosed in the present specification employs acell-free expression system such as, without limitation, prokaryoticextracts and eukaryotic extracts. Non-limiting examples of prokaryoticcell extracts include the RTS100 E. coli HY Kit (Roche Applied Science,Indianapolis, Ind.), the ActivePro In Vitro Translation Kit (Ambion,Inc., Austin, Tex.), the EcoPro™ System (EMD Biosciences-Novagen,Madison, Wis.) and the Expressway™ Plus Expression System (Invitrogen,Inc., Carlsbad, Calif.). Eukaryotic cell extract include, withoutlimitation, the RTS100 Wheat Germ CECF Kit (Roche Applied Science,Indianapolis, Ind.), the TnT® Coupled Wheat Germ Extract Systems(Promega Corp., Madison, Wis.), the Wheat Germ IVT™ Kit (Ambion, Inc.,Austin, Tex.), the Retic Lysate IVT™ Kit (Ambion, Inc., Austin, Tex.),the PROTEINscript® II System (Ambion, Inc., Austin, Tex.) and the TnT®Coupled Reticulocyte Lysate Systems (Promega Corp., Madison, Wis.).

EXAMPLES

The following non-limiting examples are provided for illustrativepurposes only in order to facilitate a more complete understanding ofdisclosed embodiments and are in no way intended to limit any of theembodiments disclosed in the present specification.

Example 1 Construction of a Modified Clostridial Toxin Comprising anAmino-Terminally Presented Enhanced Targeting Domain

This example illustrates how to make a modified Clostridial toxindisclosed in the present specification comprising an enhanced targetingdomain located at the amino terminus of the modified toxin.

1a. A Targeting-Translocation-Enzymatic Domain Organization.

A polynucleotide molecule based on BoNT/A-AP4A-GRPP (SEQ ID NO: 118)will be synthesized using standard procedures (BlueHeron® Biotechnology,Bothell, Wash.). This polynucleotide molecule encodes a BoNT/A modifiedto replace amino acids 1111-1296 of SEQ ID NO: 1, a BoNT/A H_(CC)targeting domain, with amino acids 21-50 of SEQ ID NO: 9, a GRPPtargeting domain, and has the general domain arrangement of FIG. 4A.Oligonucleotides of 20 to 50 bases in length are synthesized usingstandard phosphoramidite synthesis. These oligonucleotides will behybridized into double stranded duplexes that are ligated together toassemble the full-length polynucleotide molecule. This polynucleotidemolecule will be cloned using standard molecular biology methods into apUCBHB1 vector at the SmaI site to generate pUCBHB1/BoNT/A-AP4A-GRPP.The synthesized polynucleotide molecule is verified by sequencing usingBig Dye Terminator™ Chemistry 3.1 (Applied Biosystems, Foster City,Calif.) and an ABI 3100 sequencer (Applied Biosystems, Foster City,Calif.).

If desired, an expression optimized polynucleotide molecule based onBoNT/A-AP4A-GRPP (SEQ ID NO: 118) can be synthesized in order to improveexpression in an Escherichia coli strain. The polynucleotide moleculeencoding the BoNT/A-AP4A-GRPP will be modified to 1) contain synonymouscodons typically present in native polynucleotide molecules of anEscherichia coli strain; 2) contain a G+C content that more closelymatches the average G+C content of native polynucleotide molecules foundin an Escherichia coli strain; 3) reduce polymononucleotide regionsfound within the polynucleotide molecule; and/or 4) eliminate internalregulatory or structural sites found within the polynucleotide molecule,see, e.g., Lance E. Steward et al., Optimizing Expression of ActiveBotulinum Toxin Type E, International Patent Publication WO 2006/011966(Feb. 2, 2006); Lance E. Steward et al., Optimizing Expression of ActiveBotulinum Toxin Type A, International Patent Publication WO 2006/017749(Feb. 16, 2006). Once sequence optimization is complete,oligonucleotides of 20 to 50 bases in length are synthesized usingstandard phosphoramidite synthesis. These oligonucleotides arehybridized into double stranded duplexes that are ligated together toassemble the full-length polynucleotide molecule. This polynucleotidemolecule is cloned using standard molecular biology methods into apUCBHB1 vector at the SmaI site to generate pUCBHB1/BoNT/A-AP4A-GRPP.The synthesized polynucleotide molecule is verified by sequencing usingBig Dye Terminator™ Chemistry 3.1 (Applied Biosystems, Foster City,Calif.) and an ABI 3100 sequencer (Applied Biosystems, Foster City,Calif.). If so desired, expression optimization to a different organism,such as, e.g., a yeast strain, an insect cell-line or a mammalian cellline, can be done, see, e.g., Steward, supra, (Feb. 2, 2006); andSteward, supra, (Feb. 16, 2006).

A similar cloning strategy will be used to make pUCBHB1 cloningconstructs for BoNT/B-AP4A-GRPP, a modified BoNT/B where amino acids1098-1291 of SEQ ID NO: 2 are replaced with amino acids 21-50 of SEQ IDNO: 9; BoNT/C1-AP4A-GRPP, a modified BoNT/C1 where amino acids 1112-1291of SEQ ID NO: 3 are replaced with amino acids 21-50 of SEQ ID NO: 9;BoNT/D-AP4A-GRPP, a modified BoNT/D where amino acids 1099-1276 of SEQID NO: 4 are replaced with amino acids 21-50 of SEQ ID NO: 9;BoNT/E-AP4A-GRPP, a modified BoNT/E where amino acids 1086-1252 of SEQID NO: 5 are replaced with amino acids 21-50 of SEQ ID NO: 9;BoNT/F-AP4A-GRPP, a modified BoNT/F where amino acids 1106-1274 of SEQID NO: 6 are replaced with amino acids 21-50 of SEQ ID NO: 9;BoNT/G-AP4A-GRPP, a modified BoNT/G where amino acids 1106-1297 of SEQID NO: 7 are replaced with amino acids 21-50 of SEQ ID NO: 9; andTeNT-AP4A-GRPP, a modified TeNT where amino acids 1128-1315 of SEQ IDNO: 8 are replaced with amino acids 21-50 of SEQ ID NO: 9.

Likewise, a similar cloning strategy will be used to make pUCBHB1cloning constructs comprising a polynucleotide molecule encoding amodified Clostridial toxin-AP4A that will replace the H_(CC) targetingdomain from a Clostridial toxin the with an enhanced targeting domaincomprising, e.g, amino acids 53-81 of SEQ ID NO: 9; amino acids 53-89 ofSEQ ID NO: 9; amino acids 98-124 of SEQ ID NO: 9; amino acids 146-178 ofSEQ ID NO: 9; amino acids 132-158 of SEQ ID NO: 10; amino acids 32-58 ofSEQ ID NO: 11; amino acids 32-75 of SEQ ID NO: 11; amino acids 81-107 ofSEQ ID NO: 12; amino acids 125-151 of SEQ ID NO: 12; amino acids 81-107of SEQ ID NO: 13; amino acids 124-150 of SEQ ID NO: 13; amino acids52-78 of SEQ ID NO: 14; amino acids 52-93 of SEQ ID NO: 14; amino acids28-54 of SEQ ID NO: 15; amino acids 76-92 of SEQ ID NO: 16; amino acids59-92 of SEQ ID NO: 16; amino acids 41-50 of SEQ ID NO: 17; amino acids24-50 of SEQ ID NO: 17; amino acids 99-112 of SEQ ID NO: 18; amino acids159-193 of SEQ ID NO: 19; amino acids 154-194 of SEQ ID NO: 19; aminoacids 35-70 of SEQ ID NO: 20; amino acids 145-177 of SEQ ID NO: 20;amino acids 1-200 of SEQ ID NO: 21; amino acids 1-150 of SEQ ID NO: 22;amino acids 1-202 of SEQ ID NO: 23; amino acids 1-201 of SEQ ID NO: 24;amino acids 1-225 of SEQ ID NO: 25; amino acids 123-265 of SEQ ID NO:26; amino acids 21-153 of SEQ ID NO: 27; amino acids 57-210 of SEQ IDNO: 28; amino acids 21-99 of SEQ ID NO: 29; amino acids 31-94 of SEQ IDNO: 29; amino acids 19-178 of SEQ ID NO: 30; amino acids 1-558 of SEQ IDNO: 31; amino acids 1-371 of SEQ ID NO: 32; amino acids 49-118 of SEQ IDNO: 33; amino acids 25-180 of SEQ ID NO: 34; amino acids 1-54 of SEQ IDNO: 35; amino acids 139-257 of SEQ ID NO: 36; amino acids 129-247 of SEQID NO: 37; amino acids 19-257 of SEQ ID NO: 38; amino acids 81-210 ofSEQ ID NO: 39; amino acids 118-211 of SEQ ID NO: 40; amino acids 107-196of SEQ ID NO: 41; amino acids 96-197 of SEQ ID NO: 41; amino acids66-155 of SEQ ID NO: 42; amino acids 123-218 of SEQ ID NO: 43; aminoacids 293-390 of SEQ ID NO: 44; amino acids 317-414 of SEQ ID NO: 45;amino acids 315-412 of SEQ ID NO: 46; amino acids 276-373 of SEQ ID NO:47; amino acids 296-396 of SEQ ID NO: 48; amino acids 370-472 of SEQ IDNO: 49; amino acids 309-409 of SEQ ID NO: 50; amino acids 323-454 of SEQID NO: 51; amino acids 412-513 of SEQ ID NO: 52; amino acids 374-513 ofSEQ ID NO: 52; amino acids 330-431 of SEQ ID NO: 53; amino acids 293-431of SEQ ID NO: 53; amino acids 301-402 of SEQ ID NO: 54; amino acids323-424 of SEQ ID NO: 55; amino acids 267-372 of SEQ ID NO: 56; aminoacids 327-429 of SEQ ID NO: 57; amino acids 264-364 of SEQ ID NO: 58;amino acids 400-501 of SEQ ID NO: 59; amino acids 354-455 of SEQ ID NO:60; amino acids 352-450 of SEQ ID NO: 61; amino acids 281-375 of SEQ IDNO: 62; amino acids 376-478 of SEQ ID NO: 63; amino acids 313-407 of SEQID NO: 64; amino acids 211-308 of SEQ ID NO: 65; amino acids 321-426 ofSEQ ID NO: 66; amino acids 303-406 of SEQ ID NO: 67; amino acids 247-352of SEQ ID NO: 68; amino acids 237-352 of SEQ ID NO: 68; amino acids247-350 of SEQ ID NO: 69; amino acids 262-366 of SEQ ID NO: 70; or aminoacids 233-366 of SEQ ID NO: 70.

To construct pET29/BoNT/A-AP4A-GRPP, a pUCBHB1/BoNT/A-AP4A-GRPPconstruct will be digested with restriction endonucleases that 1) willexcise the polynucleotide molecule encoding the open reading frame ofBoNT/A-AP4A-GRPP; and 2) will enable this polynucleotide molecule to beoperably-linked to a pET29 vector (EMD Biosciences-Novagen, Madison,Wis.). This insert will be subcloned using a T4 DNA ligase procedureinto a pET29 vector that is digested with appropriate restrictionendonucleases to yield pET29/BoNT/A-AP4A-GRPP. The ligation mixture willbe transformed into chemically competent E. coli DH5α cells (Invitrogen,Inc, Carlsbad, Calif.) using a heat shock method, will be plated on 1.5%Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of Kanamycin, andwill be placed in a 37° C. incubator for overnight growth. Bacteriacontaining expression constructs will be identified as Kanamycinresistant colonies. Candidate constructs will be isolated using analkaline lysis plasmid mini-preparation procedure and will be analyzedby restriction endonuclease digest mapping to determine the presence andorientation of the insert. This cloning strategy will yield a pET29expression construct comprising the polynucleotide molecule encoding theBoNT/A-AP4A-GRPP operably-linked to a carboxyl terminal polyhistidineaffinity binding peptide.

A similar cloning strategy will be used to make pET29 expressionconstructs for other modified Clostridial toxin-AP4A-GRPP toxins, suchas, e.g., BoNT/B-AP4A-GRPP, BoNT/C1-AP4A-GRPP, BoNT/D-AP4A-GRPP,BoNT/E-AP4A-GRPP, BoNT/F-AP4A-GRPP, BoNT/G-AP4A-GRPP or TeNT-AP4A-GRPP.Likewise, a similar cloning strategy will be used to make pET29expression constructs comprising a polynucleotide molecule encoding amodified Clostridial toxin-AP4A comprising an enhanced targeting domainsuch as, e.g, amino acids 53-81 of SEQ ID NO: 9; amino acids 53-89 ofSEQ ID NO: 9; amino acids 98-124 of SEQ ID NO: 9; amino acids 146-178 ofSEQ ID NO: 9; amino acids 132-158 of SEQ ID NO: 10; amino acids 32-58 ofSEQ ID NO: 11; amino acids 32-75 of SEQ ID NO: 11; amino acids 81-107 ofSEQ ID NO: 12; amino acids 125-151 of SEQ ID NO: 12; amino acids 81-107of SEQ ID NO: 13; amino acids 124-150 of SEQ ID NO: 13; amino acids52-78 of SEQ ID NO: 14; amino acids 52-93 of SEQ ID NO: 14; amino acids28-54 of SEQ ID NO: 15; amino acids 76-92 of SEQ ID NO: 16; amino acids59-92 of SEQ ID NO: 16; amino acids 41-50 of SEQ ID NO: 17; amino acids24-50 of SEQ ID NO: 17; amino acids 99-112 of SEQ ID NO: 18; amino acids159-193 of SEQ ID NO: 19; amino acids 154-194 of SEQ ID NO: 19; aminoacids 35-70 of SEQ ID NO: 20; amino acids 145-177 of SEQ ID NO: 20;amino acids 1-200 of SEQ ID NO: 21; amino acids 1-150 of SEQ ID NO: 22;amino acids 1-202 of SEQ ID NO: 23; amino acids 1-201 of SEQ ID NO: 24;amino acids 1-225 of SEQ ID NO: 25; amino acids 123-265 of SEQ ID NO:26; amino acids 21-153 of SEQ ID NO: 27; amino acids 57-210 of SEQ IDNO: 28; amino acids 21-99 of SEQ ID NO: 29; amino acids 31-94 of SEQ IDNO: 29; amino acids 19-178 of SEQ ID NO: 30; amino acids 1-558 of SEQ IDNO: 31; amino acids 1-371 of SEQ ID NO: 32; amino acids 49-118 of SEQ IDNO: 33; amino acids 25-180 of SEQ ID NO: 34; amino acids 1-54 of SEQ IDNO: 35; amino acids 139-257 of SEQ ID NO: 36; amino acids 129-247 of SEQID NO: 37; amino acids 19-257 of SEQ ID NO: 38; amino acids 81-210 ofSEQ ID NO: 39; amino acids 118-211 of SEQ ID NO: 40; amino acids 107-196of SEQ ID NO: 41; amino acids 96-197 of SEQ ID NO: 41; amino acids66-155 of SEQ ID NO: 42; amino acids 123-218 of SEQ ID NO: 43; aminoacids 293-390 of SEQ ID NO: 44; amino acids 317-414 of SEQ ID NO: 45;amino acids 315-412 of SEQ ID NO: 46; amino acids 276-373 of SEQ ID NO:47; amino acids 296-396 of SEQ ID NO: 48; amino acids 370-472 of SEQ IDNO: 49; amino acids 309-409 of SEQ ID NO: 50; amino acids 323-454 of SEQID NO: 51; amino acids 412-513 of SEQ ID NO: 52; amino acids 374-513 ofSEQ ID NO: 52; amino acids 330-431 of SEQ ID NO: 53; amino acids 293-431of SEQ ID NO: 53; amino acids 301-402 of SEQ ID NO: 54; amino acids323-424 of SEQ ID NO: 55; amino acids 267-372 of SEQ ID NO: 56; aminoacids 327-429 of SEQ ID NO: 57; amino acids 264-364 of SEQ ID NO: 58;amino acids 400-501 of SEQ ID NO: 59; amino acids 354-455 of SEQ ID NO:60; amino acids 352-450 of SEQ ID NO: 61; amino acids 281-375 of SEQ IDNO: 62; amino acids 376-478 of SEQ ID NO: 63; amino acids 313-407 of SEQID NO: 64; amino acids 211-308 of SEQ ID NO: 65; amino acids 321-426 ofSEQ ID NO: 66; amino acids 303-406 of SEQ ID NO: 67; amino acids 247-352of SEQ ID NO: 68; amino acids 237-352 of SEQ ID NO: 68; amino acids247-350 of SEQ ID NO: 69; amino acids 262-366 of SEQ ID NO: 70; or aminoacids 233-366 of SEQ ID NO: 70.

1b. A Targeting-Enzymatic-Translocation Domain Organization.

A polynucleotide molecule based on BoNT/A-AP4B-GRPP (SEQ ID NO: 119)will be synthesized and cloned into a pUCBHB1 vector as described inExample 1a. This polynucleotide molecule encodes a BoNT/A modified toreplace amino acids 1111-1296 of SEQ ID NO: 1, a BoNT/A H_(CC) targetingdomain, with amino acids 21-50 of SEQ ID NO: 9, a GRPP targeting domain,and has the general domain arrangement of FIG. 4B. If so desired,expression optimization to a different organism, such as, e.g., abacteria, a yeast strain, an insect cell-line or a mammalian cell line,can be done as described above, see, e.g., Steward, supra, (Feb. 2,2006); and Steward, supra, (Feb. 16, 2006).

Likewise, a similar cloning strategy will be used to make pUCBHB1cloning constructs comprising a polynucleotide molecule encoding amodified Clostridial toxin-AP4B that will replace the H_(CC) targetingdomain from a Clostridial toxin the with an enhanced targeting domaincomprising, e.g, amino acids 53-81 of SEQ ID NO: 9; amino acids 53-89 ofSEQ ID NO: 9; amino acids 98-124 of SEQ ID NO: 9; amino acids 146-178 ofSEQ ID NO: 9; amino acids 132-158 of SEQ ID NO: 10; amino acids 32-58 ofSEQ ID NO: 11; amino acids 32-75 of SEQ ID NO: 11; amino acids 81-107 ofSEQ ID NO: 12; amino acids 125-151 of SEQ ID NO: 12; amino acids 81-107of SEQ ID NO: 13; amino acids 124-150 of SEQ ID NO: 13; amino acids52-78 of SEQ ID NO: 14; amino acids 52-93 of SEQ ID NO: 14; amino acids28-54 of SEQ ID NO: 15; amino acids 76-92 of SEQ ID NO: 16; amino acids59-92 of SEQ ID NO: 16; amino acids 41-50 of SEQ ID NO: 17; amino acids24-50 of SEQ ID NO: 17; amino acids 99-112 of SEQ ID NO: 18; amino acids159-193 of SEQ ID NO: 19; amino acids 154-194 of SEQ ID NO: 19; aminoacids 35-70 of SEQ ID NO: 20; amino acids 145-177 of SEQ ID NO: 20;amino acids 1-200 of SEQ ID NO: 21; amino acids 1-150 of SEQ ID NO: 22;amino acids 1-202 of SEQ ID NO: 23; amino acids 1-201 of SEQ ID NO: 24;amino acids 1-225 of SEQ ID NO: 25; amino acids 123-265 of SEQ ID NO:26; amino acids 21-153 of SEQ ID NO: 27; amino acids 57-210 of SEQ IDNO: 28; amino acids 21-99 of SEQ ID NO: 29; amino acids 31-94 of SEQ IDNO: 29; amino acids 19-178 of SEQ ID NO: 30; amino acids 1-558 of SEQ IDNO: 31; amino acids 1-371 of SEQ ID NO: 32; amino acids 49-118 of SEQ IDNO: 33; amino acids 25-180 of SEQ ID NO: 34; amino acids 1-54 of SEQ IDNO: 35; amino acids 139-257 of SEQ ID NO: 36; amino acids 129-247 of SEQID NO: 37; amino acids 19-257 of SEQ ID NO: 38; amino acids 81-210 ofSEQ ID NO: 39; amino acids 118-211 of SEQ ID NO: 40; amino acids 107-196of SEQ ID NO: 41; amino acids 96-197 of SEQ ID NO: 41; amino acids66-155 of SEQ ID NO: 42; amino acids 123-218 of SEQ ID NO: 43; aminoacids 293-390 of SEQ ID NO: 44; amino acids 317-414 of SEQ ID NO: 45;amino acids 315-412 of SEQ ID NO: 46; amino acids 276-373 of SEQ ID NO:47; amino acids 296-396 of SEQ ID NO: 48; amino acids 370-472 of SEQ IDNO: 49; amino acids 309-409 of SEQ ID NO: 50; amino acids 323-454 of SEQID NO: 51; amino acids 412-513 of SEQ ID NO: 52; amino acids 374-513 ofSEQ ID NO: 52; amino acids 330-431 of SEQ ID NO: 53; amino acids 293-431of SEQ ID NO: 53; amino acids 301-402 of SEQ ID NO: 54; amino acids323-424 of SEQ ID NO: 55; amino acids 267-372 of SEQ ID NO: 56; aminoacids 327-429 of SEQ ID NO: 57; amino acids 264-364 of SEQ ID NO: 58;amino acids 400-501 of SEQ ID NO: 59; amino acids 354-455 of SEQ ID NO:60; amino acids 352-450 of SEQ ID NO: 61; amino acids 281-375 of SEQ IDNO: 62; amino acids 376-478 of SEQ ID NO: 63; amino acids 313-407 of SEQID NO: 64; amino acids 211-308 of SEQ ID NO: 65; amino acids 321-426 ofSEQ ID NO: 66; amino acids 303-406 of SEQ ID NO: 67; amino acids 247-352of SEQ ID NO: 68; amino acids 237-352 of SEQ ID NO: 68; amino acids247-350 of SEQ ID NO: 69; amino acids 262-366 of SEQ ID NO: 70; or aminoacids 233-366 of SEQ ID NO: 70.

In addition, similar cloning strategy will be used to produce a modifiedClostridial toxin-AP4B, such as, e.g., BoNT/B-AP4B, BoNT/C1-AP4B,BoNT/D-AP4B, BoNT/E-AP4B, BoNT/F-AP4B, BoNT/G-AP4B or TeNT-AP4B, tocomprise an enhanced targeting domain comprising, e.g., amino acids53-81 of SEQ ID NO: 9; amino acids 53-89 of SEQ ID NO: 9; amino acids98-124 of SEQ ID NO: 9; amino acids 146-178 of SEQ ID NO: 9; amino acids132-158 of SEQ ID NO: 10; amino acids 32-58 of SEQ ID NO: 11; aminoacids 32-75 of SEQ ID NO: 11; amino acids 81-107 of SEQ ID NO: 12; aminoacids 125-151 of SEQ ID NO: 12; amino acids 81-107 of SEQ ID NO: 13;amino acids 124-150 of SEQ ID NO: 13; amino acids 52-78 of SEQ ID NO:14; amino acids 52-93 of SEQ ID NO: 14; amino acids 28-54 of SEQ ID NO:15; amino acids 76-92 of SEQ ID NO: 16; amino acids 59-92 of SEQ ID NO:16; amino acids 41-50 of SEQ ID NO: 17; amino acids 24-50 of SEQ ID NO:17; amino acids 99-112 of SEQ ID NO: 18; amino acids 159-193 of SEQ IDNO: 19; amino acids 154-194 of SEQ ID NO: 19; amino acids 35-70 of SEQID NO: 20; amino acids 145-177 of SEQ ID NO: 20; amino acids 1-200 ofSEQ ID NO: 21; amino acids 1-150 of SEQ ID NO: 22; amino acids 1-202 ofSEQ ID NO: 23; amino acids 1-201 of SEQ ID NO: 24; amino acids 1-225 ofSEQ ID NO: 25; amino acids 123-265 of SEQ ID NO: 26; amino acids 21-153of SEQ ID NO: 27; amino acids 57-210 of SEQ ID NO: 28; amino acids 21-99of SEQ ID NO: 29; amino acids 31-94 of SEQ ID NO: 29; amino acids 19-178of SEQ ID NO: 30; amino acids 1-558 of SEQ ID NO: 31; amino acids 1-371of SEQ ID NO: 32; amino acids 49-118 of SEQ ID NO: 33; amino acids25-180 of SEQ ID NO: 34; amino acids 1-54 of SEQ ID NO: 35; amino acids139-257 of SEQ ID NO: 36; amino acids 129-247 of SEQ ID NO: 37; aminoacids 19-257 of SEQ ID NO: 38; amino acids 81-210 of SEQ ID NO: 39;amino acids 118-211 of SEQ ID NO: 40; amino acids 107-196 of SEQ ID NO:41; amino acids 96-197 of SEQ ID NO: 41; amino acids 66-155 of SEQ IDNO: 42; amino acids 123-218 of SEQ ID NO: 43; amino acids 293-390 of SEQID NO: 44; amino acids 317-414 of SEQ ID NO: 45; amino acids 315-412 ofSEQ ID NO: 46; amino acids 276-373 of SEQ ID NO: 47; amino acids 296-396of SEQ ID NO: 48; amino acids 370-472 of SEQ ID NO: 49; amino acids309-409 of SEQ ID NO: 50; amino acids 323-454 of SEQ ID NO: 51; aminoacids 412-513 of SEQ ID NO: 52; amino acids 374-513 of SEQ ID NO: 52;amino acids 330-431 of SEQ ID NO: 53; amino acids 293-431 of SEQ ID NO:53; amino acids 301-402 of SEQ ID NO: 54; amino acids 323-424 of SEQ IDNO: 55; amino acids 267-372 of SEQ ID NO: 56; amino acids 327-429 of SEQID NO: 57; amino acids 264-364 of SEQ ID NO: 58; amino acids 400-501 ofSEQ ID NO: 59; amino acids 354-455 of SEQ ID NO: 60; amino acids 352-450of SEQ ID NO: 61; amino acids 281-375 of SEQ ID NO: 62; amino acids376-478 of SEQ ID NO: 63; amino acids 313-407 of SEQ ID NO: 64; aminoacids 211-308 of SEQ ID NO: 65; amino acids 321-426 of SEQ ID NO: 66;amino acids 303-406 of SEQ ID NO: 67; amino acids 247-352 of SEQ ID NO:68; amino acids 237-352 of SEQ ID NO: 68; amino acids 247-350 of SEQ IDNO: 69; amino acids 262-366 of SEQ ID NO: 70; or amino acids 233-366 ofSEQ ID NO: 70.

To construct pET29/BoNT/A-AP4B-GRPP, a similar cloning strategy will beused as described in Example 1a. This cloning strategy will yield apET29 expression construct comprising the polynucleotide moleculeencoding the BoNT/A-AP4B-GRPP operably-linked to a carboxyl terminalpolyhistidine affinity binding peptide. A similar cloning strategy willbe used to make pET29 expression constructs for other modifiedClostridial toxin-AP4B-GRPP toxins, such as, e.g., BoNT/B-AP4B-GRPP,BoNT/C1-AP4B-GRPP, BoNT/D-AP4B-GRPP, BoNT/E-AP4B-GRPP, BoNT/F-AP4B-GRPP,BoNT/G-AP4B-GRPP or TeNT-AP4B-GRPP. Likewise, a similar cloning strategywill be used to make pET29 expression constructs comprising apolynucleotide molecule encoding a modified Clostridial toxin-AP4Bcomprising an enhanced targeting domain such as, e.g, amino acids 53-81of SEQ ID NO: 9; amino acids 53-89 of SEQ ID NO: 9; amino acids 98-124of SEQ ID NO: 9; amino acids 146-178 of SEQ ID NO: 9; amino acids132-158 of SEQ ID NO: 10; amino acids 32-58 of SEQ ID NO: 11; aminoacids 32-75 of SEQ ID NO: 11; amino acids 81-107 of SEQ ID NO: 12; aminoacids 125-151 of SEQ ID NO: 12; amino acids 81-107 of SEQ ID NO: 13;amino acids 124-150 of SEQ ID NO: 13; amino acids 52-78 of SEQ ID NO:14; amino acids 52-93 of SEQ ID NO: 14; amino acids 28-54 of SEQ ID NO:15; amino acids 76-92 of SEQ ID NO: 16; amino acids 59-92 of SEQ ID NO:16; amino acids 41-50 of SEQ ID NO: 17; amino acids 24-50 of SEQ ID NO:17; amino acids 99-112 of SEQ ID NO: 18; amino acids 159-193 of SEQ IDNO: 19; amino acids 154-194 of SEQ ID NO: 19; amino acids 35-70 of SEQID NO: 20; amino acids 145-177 of SEQ ID NO: 20; amino acids 1-200 ofSEQ ID NO: 21; amino acids 1-150 of SEQ ID NO: 22; amino acids 1-202 ofSEQ ID NO: 23; amino acids 1-201 of SEQ ID NO: 24; amino acids 1-225 ofSEQ ID NO: 25; amino acids 123-265 of SEQ ID NO: 26; amino acids 21-153of SEQ ID NO: 27; amino acids 57-210 of SEQ ID NO: 28; amino acids 21-99of SEQ ID NO: 29; amino acids 31-94 of SEQ ID NO: 29; amino acids 19-178of SEQ ID NO: 30; amino acids 1-558 of SEQ ID NO: 31; amino acids 1-371of SEQ ID NO: 32; amino acids 49-118 of SEQ ID NO: 33; amino acids25-180 of SEQ ID NO: 34; amino acids 1-54 of SEQ ID NO: 35; amino acids139-257 of SEQ ID NO: 36; amino acids 129-247 of SEQ ID NO: 37; aminoacids 19-257 of SEQ ID NO: 38; amino acids 81-210 of SEQ ID NO: 39;amino acids 118-211 of SEQ ID NO: 40; amino acids 107-196 of SEQ ID NO:41; amino acids 96-197 of SEQ ID NO: 41; amino acids 66-155 of SEQ IDNO: 42; amino acids 123-218 of SEQ ID NO: 43; amino acids 293-390 of SEQID NO: 44; amino acids 317-414 of SEQ ID NO: 45; amino acids 315-412 ofSEQ ID NO: 46; amino acids 276-373 of SEQ ID NO: 47; amino acids 296-396of SEQ ID NO: 48; amino acids 370-472 of SEQ ID NO: 49; amino acids309-409 of SEQ ID NO: 50; amino acids 323-454 of SEQ ID NO: 51; aminoacids 412-513 of SEQ ID NO: 52; amino acids 374-513 of SEQ ID NO: 52;amino acids 330-431 of SEQ ID NO: 53; amino acids 293-431 of SEQ ID NO:53; amino acids 301-402 of SEQ ID NO: 54; amino acids 323-424 of SEQ IDNO: 55; amino acids 267-372 of SEQ ID NO: 56; amino acids 327-429 of SEQID NO: 57; amino acids 264-364 of SEQ ID NO: 58; amino acids 400-501 ofSEQ ID NO: 59; amino acids 354-455 of SEQ ID NO: 60; amino acids 352-450of SEQ ID NO: 61; amino acids 281-375 of SEQ ID NO: 62; amino acids376-478 of SEQ ID NO: 63; amino acids 313-407 of SEQ ID NO: 64; aminoacids 211-308 of SEQ ID NO: 65; amino acids 321-426 of SEQ ID NO: 66;amino acids 303-406 of SEQ ID NO: 67; amino acids 247-352 of SEQ ID NO:68; amino acids 237-352 of SEQ ID NO: 68; amino acids 247-350 of SEQ IDNO: 69; amino acids 262-366 of SEQ ID NO: 70; or amino acids 233-366 ofSEQ ID NO: 70.

Example 2 Construction of a Modified Clostridial Toxin Comprising aCentrally Presented Enhanced Targeting Domain

This example illustrates how to make a modified Clostridial toxindisclosed in the present specification comprising an enhanced targetingdomain located between two other domains of the modified toxin.

2a. An Enzymatic-Targeting-Translocation Domain Organization.

A polynucleotide molecule based on BoNT/A-CP5A-GRPP (SEQ ID NO: 196)will be synthesized using standard procedures (BlueHeron® Biotechnology,Bothell, Wash.). This polynucleotide molecule encodes a BoNT/A modifiedto replace amino acids 1111-1296 of SEQ ID NO: 1, a BoNT/A H_(CC)targeting domain, with amino acids 21-50 of SEQ ID NO: 9, a GRPPtargeting domain, and has the general domain arrangement of FIG. 5A.Cleavage of an enterokinase cleavage site used to form the di-chaintoxin also exposes the first amino acid of the GRPP targeting domain.Oligonucleotides of 20 to 50 bases in length are synthesized usingstandard phosphoramidite synthesis. These oligonucleotides will behybridized into double stranded duplexes that are ligated together toassemble the full-length polynucleotide molecule. This polynucleotidemolecule will be cloned using standard molecular biology methods into apUCBHB1 vector at the SmaI site to generate pUCBHB1/BoNT/A-CP5A-GRPP.The synthesized polynucleotide molecule is verified by sequencing usingBig Dye Terminator™ Chemistry 3.1 (Applied Biosystems, Foster City,Calif.) and an ABI 3100 sequencer (Applied Biosystems, Foster City,Calif.).

If desired, an expression optimized polynucleotide molecule based onBoNT/A-CP5A-GRPP (SEQ ID NO: 196) can be synthesized in order to improveexpression in an Escherichia coli strain. The polynucleotide moleculeencoding the BoNT/A-CP5A-GRPP will be modified to 1) contain synonymouscodons typically present in native polynucleotide molecules of anEscherichia coli strain; 2) contain a G+C content that more closelymatches the average G+C content of native polynucleotide molecules foundin an Escherichia coli strain; 3) reduce polymononucleotide regionsfound within the polynucleotide molecule; and/or 4) eliminate internalregulatory or structural sites found within the polynucleotide molecule,see, e.g., Lance E. Steward et al., Optimizing Expression of ActiveBotulinum Toxin Type E, International Patent Publication WO 2006/011966(Feb. 2, 2006); Lance E. Steward et al., Optimizing Expression of ActiveBotulinum Toxin Type A, International Patent Publication WO 2006/017749(Feb. 16, 2006). Once sequence optimization is complete,oligonucleotides of 20 to 50 bases in length are synthesized usingstandard phosphoramidite synthesis. These oligonucleotides arehybridized into double stranded duplexes that are ligated together toassemble the full-length polynucleotide molecule. This polynucleotidemolecule is cloned using standard molecular biology methods into apUCBHB1 vector at the SmaI site to generate pUCBHB1/BoNT/A-CP5A-GRPP.The synthesized polynucleotide molecule is verified by sequencing usingBig Dye Terminator™ Chemistry 3.1 (CP5 Applied Biosystems, Foster City,Calif.) and an ABI 3100 sequencer (CP5 Applied Biosystems, Foster City,Calif.). If so desired, expression optimization to a different organism,such as, e.g., a yeast strain, an insect cell-line or a mammalian cellline, can be done, see, e.g., Steward, supra, (Feb. 2, 2006); andSteward, supra, (Feb. 16, 2006).

A similar cloning strategy will be used to make pUCBHB1 cloningconstructs for BoNT/B-CP5A-GRPP, a modified BoNT/B where amino acids1098-1291 of SEQ ID NO: 2 are replaced with amino acids 21-50 of SEQ IDNO: 9; BoNT/C1-CP5A-GRPP, a modified BoNT/C1 where amino acids 1112-1291of SEQ ID NO: 3 are replaced with amino acids 21-50 of SEQ ID NO: 9;BoNT/D-CP5A-GRPP, a modified BoNT/D where amino acids 1099-1276 of SEQID NO: 4 are replaced with amino acids 21-50 of SEQ ID NO: 9;BoNT/E-CP5A-GRPP, a modified BoNT/E where amino acids 1086-1252 of SEQID NO: 5 are replaced with amino acids 21-50 of SEQ ID NO: 9;BoNT/F-CP5A-GRPP, a modified BoNT/F where amino acids 1106-1274 of SEQID NO: 6 are replaced with amino acids 21-50 of SEQ ID NO: 9;BoNT/G-CP5A-GRPP, a modified BoNT/G where amino acids 1106-1297 of SEQID NO: 7 are replaced with amino acids 21-50 of SEQ ID NO: 9; andTeNT-CP5A-GRPP, a modified TeNT where amino acids 1128-1315 of SEQ IDNO: 8 are replaced with amino acids 21-50 of SEQ ID NO: 9.

Likewise, a similar cloning strategy will be used to make pUCBHB1cloning constructs comprising a polynucleotide molecule encoding amodified Clostridial toxin-CP5A that will replace the H_(CC) targetingdomain from a Clostridial toxin the with an enhanced targeting domaincomprising, e.g, amino acids 53-81 of SEQ ID NO: 9; amino acids 53-89 ofSEQ ID NO: 9; amino acids 98-124 of SEQ ID NO: 9; amino acids 146-178 ofSEQ ID NO: 9; amino acids 132-158 of SEQ ID NO: 10; amino acids 32-58 ofSEQ ID NO: 11; amino acids 32-75 of SEQ ID NO: 11; amino acids 81-107 ofSEQ ID NO: 12; amino acids 125-151 of SEQ ID NO: 12; amino acids 81-107of SEQ ID NO: 13; amino acids 124-150 of SEQ ID NO: 13; amino acids52-78 of SEQ ID NO: 14; amino acids 52-93 of SEQ ID NO: 14; amino acids28-54 of SEQ ID NO: 15; amino acids 76-92 of SEQ ID NO: 16; amino acids59-92 of SEQ ID NO: 16; amino acids 41-50 of SEQ ID NO: 17; amino acids24-50 of SEQ ID NO: 17; amino acids 99-112 of SEQ ID NO: 18; amino acids159-193 of SEQ ID NO: 19; amino acids 154-194 of SEQ ID NO: 19; aminoacids 35-70 of SEQ ID NO: 20; amino acids 145-177 of SEQ ID NO: 20;amino acids 1-200 of SEQ ID NO: 21; amino acids 1-150 of SEQ ID NO: 22;amino acids 1-202 of SEQ ID NO: 23; amino acids 1-201 of SEQ ID NO: 24;amino acids 1-225 of SEQ ID NO: 25; amino acids 123-265 of SEQ ID NO:26; amino acids 21-153 of SEQ ID NO: 27; amino acids 57-210 of SEQ IDNO: 28; amino acids 21-99 of SEQ ID NO: 29; amino acids 31-94 of SEQ IDNO: 29; amino acids 19-178 of SEQ ID NO: 30; amino acids 1-558 of SEQ IDNO: 31; amino acids 1-371 of SEQ ID NO: 32; amino acids 49-118 of SEQ IDNO: 33; amino acids 25-180 of SEQ ID NO: 34; amino acids 1-54 of SEQ IDNO: 35; amino acids 139-257 of SEQ ID NO: 36; amino acids 129-247 of SEQID NO: 37; amino acids 19-257 of SEQ ID NO: 38; amino acids 81-210 ofSEQ ID NO: 39; amino acids 118-211 of SEQ ID NO: 40; amino acids 107-196of SEQ ID NO: 41; amino acids 96-197 of SEQ ID NO: 41; amino acids66-155 of SEQ ID NO: 42; amino acids 123-218 of SEQ ID NO: 43; aminoacids 293-390 of SEQ ID NO: 44; amino acids 317-414 of SEQ ID NO: 45;amino acids 315-412 of SEQ ID NO: 46; amino acids 276-373 of SEQ ID NO:47; amino acids 296-396 of SEQ ID NO: 48; amino acids 370-472 of SEQ IDNO: 49; amino acids 309-409 of SEQ ID NO: 50; amino acids 323-454 of SEQID NO: 51; amino acids 412-513 of SEQ ID NO: 52; amino acids 374-513 ofSEQ ID NO: 52; amino acids 330-431 of SEQ ID NO: 53; amino acids 293-431of SEQ ID NO: 53; amino acids 301-402 of SEQ ID NO: 54; amino acids323-424 of SEQ ID NO: 55; amino acids 267-372 of SEQ ID NO: 56; aminoacids 327-429 of SEQ ID NO: 57; amino acids 264-364 of SEQ ID NO: 58;amino acids 400-501 of SEQ ID NO: 59; amino acids 354-455 of SEQ ID NO:60; amino acids 352-450 of SEQ ID NO: 61; amino acids 281-375 of SEQ IDNO: 62; amino acids 376-478 of SEQ ID NO: 63; amino acids 313-407 of SEQID NO: 64; amino acids 211-308 of SEQ ID NO: 65; amino acids 321-426 ofSEQ ID NO: 66; amino acids 303-406 of SEQ ID NO: 67; amino acids 247-352of SEQ ID NO: 68; amino acids 237-352 of SEQ ID NO: 68; amino acids247-350 of SEQ ID NO: 69; amino acids 262-366 of SEQ ID NO: 70; or aminoacids 233-366 of SEQ ID NO: 70.

To construct pET29/BoNT/A-CP5A-GRPP, a pUCBHB1/BoNT/A-CP5A-GRPPconstruct will be digested with restriction endonucleases that 1) willexcise the polynucleotide molecule encoding the open reading frame ofBoNT/A-CP5A-GRPP; and 2) will enable this polynucleotide molecule to beoperably-linked to a pET29 vector (EMD Biosciences-Novagen, Madison,Wis.). This insert will be subcloned using a T4 DNA ligase procedureinto a pET29 vector that is digested with appropriate restrictionendonucleases to yield pET29/BoNT/A-CP5A-GRPP. The ligation mixture willbe transformed into chemically competent E. coli DH5α cells (Invitrogen,Inc, Carlsbad, Calif.) using a heat shock method, will be plated on 1.5%Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of Kanamycin, andwill be placed in a 37° C. incubator for overnight growth. Bacteriacontaining expression constructs will be identified as Kanamycinresistant colonies. Candidate constructs will be isolated using analkaline lysis plasmid mini-preparation procedure and will be analyzedby restriction endonuclease digest mapping to determine the presence andorientation of the insert. This cloning strategy will yield a pET29expression construct comprising the polynucleotide molecule encoding theBoNT/A-CP5A-GRPP operably-linked to a carboxyl terminal polyhistidineaffinity binding peptide.

A similar cloning strategy will be used to make pET29 expressionconstructs for other modified Clostridial toxin-CP5A-GRPP toxins, suchas, e.g., BoNT/B-CP5A-GRPP, BoNT/C1-CP5A-GRPP, BoNT/D-CP5A-GRPP,BoNT/E-CP5A-GRPP, BoNT/F-CP5A-GRPP, BoNT/G-CP5A-GRPP or TeNT-CP5A-GRPP.Likewise, a similar cloning strategy will be used to make pET29expression constructs comprising a polynucleotide molecule encoding amodified Clostridial toxin-CP5A comprising an enhanced targeting domainsuch as, e.g, amino acids 53-81 of SEQ ID NO: 9; amino acids 53-89 ofSEQ ID NO: 9; amino acids 98-124 of SEQ ID NO: 9; amino acids 146-178 ofSEQ ID NO: 9; amino acids 132-158 of SEQ ID NO: 10; amino acids 32-58 ofSEQ ID NO: 11; amino acids 32-75 of SEQ ID NO: 11; amino acids 81-107 ofSEQ ID NO: 12; amino acids 125-151 of SEQ ID NO: 12; amino acids 81-107of SEQ ID NO: 13; amino acids 124-150 of SEQ ID NO: 13; amino acids52-78 of SEQ ID NO: 14; amino acids 52-93 of SEQ ID NO: 14; amino acids28-54 of SEQ ID NO: 15; amino acids 76-92 of SEQ ID NO: 16; amino acids59-92 of SEQ ID NO: 16; amino acids 41-50 of SEQ ID NO: 17; amino acids24-50 of SEQ ID NO: 17; amino acids 99-112 of SEQ ID NO: 18; amino acids159-193 of SEQ ID NO: 19; amino acids 154-194 of SEQ ID NO: 19; aminoacids 35-70 of SEQ ID NO: 20; amino acids 145-177 of SEQ ID NO: 20;amino acids 1-200 of SEQ ID NO: 21; amino acids 1-150 of SEQ ID NO: 22;amino acids 1-202 of SEQ ID NO: 23; amino acids 1-201 of SEQ ID NO: 24;amino acids 1-225 of SEQ ID NO: 25; amino acids 123-265 of SEQ ID NO:26; amino acids 21-153 of SEQ ID NO: 27; amino acids 57-210 of SEQ IDNO: 28; amino acids 21-99 of SEQ ID NO: 29; amino acids 31-94 of SEQ IDNO: 29; amino acids 19-178 of SEQ ID NO: 30; amino acids 1-558 of SEQ IDNO: 31; amino acids 1-371 of SEQ ID NO: 32; amino acids 49-118 of SEQ IDNO: 33; amino acids 25-180 of SEQ ID NO: 34; amino acids 1-54 of SEQ IDNO: 35; amino acids 139-257 of SEQ ID NO: 36; amino acids 129-247 of SEQID NO: 37; amino acids 19-257 of SEQ ID NO: 38; amino acids 81-210 ofSEQ ID NO: 39; amino acids 118-211 of SEQ ID NO: 40; amino acids 107-196of SEQ ID NO: 41; amino acids 96-197 of SEQ ID NO: 41; amino acids66-155 of SEQ ID NO: 42; amino acids 123-218 of SEQ ID NO: 43; aminoacids 293-390 of SEQ ID NO: 44; amino acids 317-414 of SEQ ID NO: 45;amino acids 315-412 of SEQ ID NO: 46; amino acids 276-373 of SEQ ID NO:47; amino acids 296-396 of SEQ ID NO: 48; amino acids 370-472 of SEQ IDNO: 49; amino acids 309-409 of SEQ ID NO: 50; amino acids 323-454 of SEQID NO: 51; amino acids 412-513 of SEQ ID NO: 52; amino acids 374-513 ofSEQ ID NO: 52; amino acids 330-431 of SEQ ID NO: 53; amino acids 293-431of SEQ ID NO: 53; amino acids 301-402 of SEQ ID NO: 54; amino acids323-424 of SEQ ID NO: 55; amino acids 267-372 of SEQ ID NO: 56; aminoacids 327-429 of SEQ ID NO: 57; amino acids 264-364 of SEQ ID NO: 58;amino acids 400-501 of SEQ ID NO: 59; amino acids 354-455 of SEQ ID NO:60; amino acids 352-450 of SEQ ID NO: 61; amino acids 281-375 of SEQ IDNO: 62; amino acids 376-478 of SEQ ID NO: 63; amino acids 313-407 of SEQID NO: 64; amino acids 211-308 of SEQ ID NO: 65; amino acids 321-426 ofSEQ ID NO: 66; amino acids 303-406 of SEQ ID NO: 67; amino acids 247-352of SEQ ID NO: 68; amino acids 237-352 of SEQ ID NO: 68; amino acids247-350 of SEQ ID NO: 69; amino acids 262-366 of SEQ ID NO: 70; or aminoacids 233-366 of SEQ ID NO: 70.

2b. A Translocation-Targeting-Enzymatic Domain Organization.

A polynucleotide molecule based on BoNT/A-CP5B-GRPP (SEQ ID NO: 120)will be synthesized and cloned into a pUCBHB1 vector as described inExample 1a. This polynucleotide molecule encodes a BoNT/A modified toreplace amino acids 1111-1296 of SEQ ID NO: 1, a BoNT/A H_(CC) targetingdomain, with amino acids 21-50 of SEQ ID NO: 9, a GRPP targeting domain,and has the general domain arrangement of FIG. 5B. Cleavage of anenterokinase cleavage site used to form the di-chain toxin also exposesthe first amino acid of the GRPP targeting domain. If so desired,expression optimization to a different organism, such as, e.g., abacteria, a yeast strain, an insect cell-line or a mammalian cell line,can be done as described above, see, e.g., Steward, supra, (Feb. 2,2006); and Steward, supra, (Feb. 16, 2006).

Likewise, a similar cloning strategy will be used to make pUCBHB1cloning constructs comprising a polynucleotide molecule encoding amodified Clostridial toxin-CP5B that will replace the H_(CC) targetingdomain from a Clostridial toxin the with an enhanced targeting domaincomprising, e.g, amino acids 53-81 of SEQ ID NO: 9; amino acids 53-89 ofSEQ ID NO: 9; amino acids 98-124 of SEQ ID NO: 9; amino acids 146-178 ofSEQ ID NO: 9; amino acids 132-158 of SEQ ID NO: 10; amino acids 32-58 ofSEQ ID NO: 11; amino acids 32-75 of SEQ ID NO: 11; amino acids 81-107 ofSEQ ID NO: 12; amino acids 125-151 of SEQ ID NO: 12; amino acids 81-107of SEQ ID NO: 13; amino acids 124-150 of SEQ ID NO: 13; amino acids52-78 of SEQ ID NO: 14; amino acids 52-93 of SEQ ID NO: 14; amino acids28-54 of SEQ ID NO: 15; amino acids 76-92 of SEQ ID NO: 16; amino acids59-92 of SEQ ID NO: 16; amino acids 41-50 of SEQ ID NO: 17; amino acids24-50 of SEQ ID NO: 17; amino acids 99-112 of SEQ ID NO: 18; amino acids159-193 of SEQ ID NO: 19; amino acids 154-194 of SEQ ID NO: 19; aminoacids 35-70 of SEQ ID NO: 20; amino acids 145-177 of SEQ ID NO: 20;amino acids 1-200 of SEQ ID NO: 21; amino acids 1-150 of SEQ ID NO: 22;amino acids 1-202 of SEQ ID NO: 23; amino acids 1-201 of SEQ ID NO: 24;amino acids 1-225 of SEQ ID NO: 25; amino acids 123-265 of SEQ ID NO:26; amino acids 21-153 of SEQ ID NO: 27; amino acids 57-210 of SEQ IDNO: 28; amino acids 21-99 of SEQ ID NO: 29; amino acids 31-94 of SEQ IDNO: 29; amino acids 19-178 of SEQ ID NO: 30; amino acids 1-558 of SEQ IDNO: 31; amino acids 1-371 of SEQ ID NO: 32; amino acids 49-118 of SEQ IDNO: 33; amino acids 25-180 of SEQ ID NO: 34; amino acids 1-54 of SEQ IDNO: 35; amino acids 139-257 of SEQ ID NO: 36; amino acids 129-247 of SEQID NO: 37; amino acids 19-257 of SEQ ID NO: 38; amino acids 81-210 ofSEQ ID NO: 39; amino acids 118-211 of SEQ ID NO: 40; amino acids 107-196of SEQ ID NO: 41; amino acids 96-197 of SEQ ID NO: 41; amino acids66-155 of SEQ ID NO: 42; amino acids 123-218 of SEQ ID NO: 43; aminoacids 293-390 of SEQ ID NO: 44; amino acids 317-414 of SEQ ID NO: 45;amino acids 315-412 of SEQ ID NO: 46; amino acids 276-373 of SEQ ID NO:47; amino acids 296-396 of SEQ ID NO: 48; amino acids 370-472 of SEQ IDNO: 49; amino acids 309-409 of SEQ ID NO: 50; amino acids 323-454 of SEQID NO: 51; amino acids 412-513 of SEQ ID NO: 52; amino acids 374-513 ofSEQ ID NO: 52; amino acids 330-431 of SEQ ID NO: 53; amino acids 293-431of SEQ ID NO: 53; amino acids 301-402 of SEQ ID NO: 54; amino acids323-424 of SEQ ID NO: 55; amino acids 267-372 of SEQ ID NO: 56; aminoacids 327-429 of SEQ ID NO: 57; amino acids 264-364 of SEQ ID NO: 58;amino acids 400-501 of SEQ ID NO: 59; amino acids 354-455 of SEQ ID NO:60; amino acids 352-450 of SEQ ID NO: 61; amino acids 281-375 of SEQ IDNO: 62; amino acids 376-478 of SEQ ID NO: 63; amino acids 313-407 of SEQID NO: 64; amino acids 211-308 of SEQ ID NO: 65; amino acids 321-426 ofSEQ ID NO: 66; amino acids 303-406 of SEQ ID NO: 67; amino acids 247-352of SEQ ID NO: 68; amino acids 237-352 of SEQ ID NO: 68; amino acids247-350 of SEQ ID NO: 69; amino acids 262-366 of SEQ ID NO: 70; or aminoacids 233-366 of SEQ ID NO: 70.

In addition, similar cloning strategy will be used to produce a modifiedClostridial toxin-XP6B, such as, e.g., BoNT/B-CP5B, BoNT/C1-CP5B,BoNT/D-CP5B, BoNT/E-CP5B, BoNT/F-CP5B, BoNT/G-CP5B or TeNT-CP5B, tocomprise an enhanced targeting domain comprising, e.g., amino acids53-81 of SEQ ID NO: 9; amino acids 53-89 of SEQ ID NO: 9; amino acids98-124 of SEQ ID NO: 9; amino acids 146-178 of SEQ ID NO: 9; amino acids132-158 of SEQ ID NO: 10; amino acids 32-58 of SEQ ID NO: 11; aminoacids 32-75 of SEQ ID NO: 11; amino acids 81-107 of SEQ ID NO: 12; aminoacids 125-151 of SEQ ID NO: 12; amino acids 81-107 of SEQ ID NO: 13;amino acids 124-150 of SEQ ID NO: 13; amino acids 52-78 of SEQ ID NO:14; amino acids 52-93 of SEQ ID NO: 14; amino acids 28-54 of SEQ ID NO:15; amino acids 76-92 of SEQ ID NO: 16; amino acids 59-92 of SEQ ID NO:16; amino acids 41-50 of SEQ ID NO: 17; amino acids 24-50 of SEQ ID NO:17; amino acids 99-112 of SEQ ID NO: 18; amino acids 159-193 of SEQ IDNO: 19; amino acids 154-194 of SEQ ID NO: 19; amino acids 35-70 of SEQID NO: 20; amino acids 145-177 of SEQ ID NO: 20; amino acids 1-200 ofSEQ ID NO: 21; amino acids 1-150 of SEQ ID NO: 22; amino acids 1-202 ofSEQ ID NO: 23; amino acids 1-201 of SEQ ID NO: 24; amino acids 1-225 ofSEQ ID NO: 25; amino acids 123-265 of SEQ ID NO: 26; amino acids 21-153of SEQ ID NO: 27; amino acids 57-210 of SEQ ID NO: 28; amino acids 21-99of SEQ ID NO: 29; amino acids 31-94 of SEQ ID NO: 29; amino acids 19-178of SEQ ID NO: 30; amino acids 1-558 of SEQ ID NO: 31; amino acids 1-371of SEQ ID NO: 32; amino acids 49-118 of SEQ ID NO: 33; amino acids25-180 of SEQ ID NO: 34; amino acids 1-54 of SEQ ID NO: 35; amino acids139-257 of SEQ ID NO: 36; amino acids 129-247 of SEQ ID NO: 37; aminoacids 19-257 of SEQ ID NO: 38; amino acids 81-210 of SEQ ID NO: 39;amino acids 118-211 of SEQ ID NO: 40; amino acids 107-196 of SEQ ID NO:41; amino acids 96-197 of SEQ ID NO: 41; amino acids 66-155 of SEQ IDNO: 42; amino acids 123-218 of SEQ ID NO: 43; amino acids 293-390 of SEQID NO: 44; amino acids 317-414 of SEQ ID NO: 45; amino acids 315-412 ofSEQ ID NO: 46; amino acids 276-373 of SEQ ID NO: 47; amino acids 296-396of SEQ ID NO: 48; amino acids 370-472 of SEQ ID NO: 49; amino acids309-409 of SEQ ID NO: 50; amino acids 323-454 of SEQ ID NO: 51; aminoacids 412-513 of SEQ ID NO: 52; amino acids 374-513 of SEQ ID NO: 52;amino acids 330-431 of SEQ ID NO: 53; amino acids 293-431 of SEQ ID NO:53; amino acids 301-402 of SEQ ID NO: 54; amino acids 323-424 of SEQ IDNO: 55; amino acids 267-372 of SEQ ID NO: 56; amino acids 327-429 of SEQID NO: 57; amino acids 264-364 of SEQ ID NO: 58; amino acids 400-501 ofSEQ ID NO: 59; amino acids 354-455 of SEQ ID NO: 60; amino acids 352-450of SEQ ID NO: 61; amino acids 281-375 of SEQ ID NO: 62; amino acids376-478 of SEQ ID NO: 63; amino acids 313-407 of SEQ ID NO: 64; aminoacids 211-308 of SEQ ID NO: 65; amino acids 321-426 of SEQ ID NO: 66;amino acids 303-406 of SEQ ID NO: 67; amino acids 247-352 of SEQ ID NO:68; amino acids 237-352 of SEQ ID NO: 68; amino acids 247-350 of SEQ IDNO: 69; amino acids 262-366 of SEQ ID NO: 70; or amino acids 233-366 ofSEQ ID NO: 70.

To construct pET29/BoNT/A-CP5B-GRPP, a similar cloning strategy will beused as described in Example 1a. This cloning strategy will yield apET29 expression construct comprising the polynucleotide moleculeencoding the BoNT/A-CP5B-GRPP operably-linked to a carboxyl terminalpolyhistidine affinity binding peptide. A similar cloning strategy willbe used to make pET29 expression constructs for other modifiedClostridial toxin-CP5B-GRPP toxins, such as, e.g., BoNT/B-CP5B-GRPP,BoNT/C1-CP5B-GRPP, BoNT/D-CP5B-GRPP, BoNT/E-CP5B-GRPP, BoNT/F-CP5B-GRPP,BoNT/G-CP5B-GRPP or TeNT-CP5B-GRPP. Likewise, a similar cloning strategywill be used to make pET29 expression constructs comprising apolynucleotide molecule encoding a modified Clostridial toxin-CP5B withan enhanced targeting domain comprising, e.g, amino acids 53-81 of SEQID NO: 9; amino acids 53-89 of SEQ ID NO: 9; amino acids 98-124 of SEQID NO: 9; amino acids 146-178 of SEQ ID NO: 9; amino acids 132-158 ofSEQ ID NO: 10; amino acids 32-58 of SEQ ID NO: 11; amino acids 32-75 ofSEQ ID NO: 11; amino acids 81-107 of SEQ ID NO: 12; amino acids 125-151of SEQ ID NO: 12; amino acids 81-107 of SEQ ID NO: 13; amino acids124-150 of SEQ ID NO: 13; amino acids 52-78 of SEQ ID NO: 14; aminoacids 52-93 of SEQ ID NO: 14; amino acids 28-54 of SEQ ID NO: 15; aminoacids 76-92 of SEQ ID NO: 16; amino acids 59-92 of SEQ ID NO: 16; aminoacids 41-50 of SEQ ID NO: 17; amino acids 24-50 of SEQ ID NO: 17; aminoacids 99-112 of SEQ ID NO: 18; amino acids 159-193 of SEQ ID NO: 19;amino acids 154-194 of SEQ ID NO: 19; amino acids 35-70 of SEQ ID NO:20; amino acids 145-177 of SEQ ID NO: 20; amino acids 1-200 of SEQ IDNO: 21; amino acids 1-150 of SEQ ID NO: 22; amino acids 1-202 of SEQ IDNO: 23; amino acids 1-201 of SEQ ID NO: 24; amino acids 1-225 of SEQ IDNO: 25; amino acids 123-265 of SEQ ID NO: 26; amino acids 21-153 of SEQID NO: 27; amino acids 57-210 of SEQ ID NO: 28; amino acids 21-99 of SEQID NO: 29; amino acids 31-94 of SEQ ID NO: 29; amino acids 19-178 of SEQID NO: 30; amino acids 1-558 of SEQ ID NO: 31; amino acids 1-371 of SEQID NO: 32; amino acids 49-118 of SEQ ID NO: 33; amino acids 25-180 ofSEQ ID NO: 34; amino acids 1-54 of SEQ ID NO: 35; amino acids 139-257 ofSEQ ID NO: 36; amino acids 129-247 of SEQ ID NO: 37; amino acids 19-257of SEQ ID NO: 38; amino acids 81-210 of SEQ ID NO: 39; amino acids118-211 of SEQ ID NO: 40; amino acids 107-196 of SEQ ID NO: 41; aminoacids 96-197 of SEQ ID NO: 41; amino acids 66-155 of SEQ ID NO: 42;amino acids 123-218 of SEQ ID NO: 43; amino acids 293-390 of SEQ ID NO:44; amino acids 317-414 of SEQ ID NO: 45; amino acids 315-412 of SEQ IDNO: 46; amino acids 276-373 of SEQ ID NO: 47; amino acids 296-396 of SEQID NO: 48; amino acids 370-472 of SEQ ID NO: 49; amino acids 309-409 ofSEQ ID NO: 50; amino acids 323-454 of SEQ ID NO: 51; amino acids 412-513of SEQ ID NO: 52; amino acids 374-513 of SEQ ID NO: 52; amino acids330-431 of SEQ ID NO: 53; amino acids 293-431 of SEQ ID NO: 53; aminoacids 301-402 of SEQ ID NO: 54; amino acids 323-424 of SEQ ID NO: 55;amino acids 267-372 of SEQ ID NO: 56; amino acids 327-429 of SEQ ID NO:57; amino acids 264-364 of SEQ ID NO: 58; amino acids 400-501 of SEQ IDNO: 59; amino acids 354-455 of SEQ ID NO: 60; amino acids 352-450 of SEQID NO: 61; amino acids 281-375 of SEQ ID NO: 62; amino acids 376-478 ofSEQ ID NO: 63; amino acids 313-407 of SEQ ID NO: 64; amino acids 211-308of SEQ ID NO: 65; amino acids 321-426 of SEQ ID NO: 66; amino acids303-406 of SEQ ID NO: 67; amino acids 247-352 of SEQ ID NO: 68; aminoacids 237-352 of SEQ ID NO: 68; amino acids 247-350 of SEQ ID NO: 69;amino acids 262-366 of SEQ ID NO: 70; or amino acids 233-366 of SEQ IDNO: 70.

Example 3 Construction of a Modified Clostridial Toxin Comprising aCarboxyl-Terminally Presented Enhanced Targeting Domain

This example illustrates how to make a modified Clostridial toxindisclosed in the present specification comprising an enhanced targetingdomain located at the carboxyl terminus of the modified toxin.

3a. An Enzymatic-Translocation-Targeting Domain Organization.

A polynucleotide molecule based on BoNT/A-XP6A-GRPP (SEQ ID NO: 121)will be synthesized using standard procedures (BlueHeron® Biotechnology,Bothell, Wash.). This polynucleotide molecule encodes a BoNT/A modifiedto replace amino acids 1111-1296 of SEQ ID NO: 1, a BoNT/A H_(CC)targeting domain, with amino acids 21-50 of SEQ ID NO: 9, a GRPPtargeting domain, and has the general domain arrangement of FIG. 6A.Oligonucleotides of 20 to 50 bases in length are synthesized usingstandard phosphoramidite synthesis. These oligonucleotides will behybridized into double stranded duplexes that are ligated together toassemble the full-length polynucleotide molecule. This polynucleotidemolecule will be cloned using standard molecular biology methods into apUCBHB1 vector at the SmaI site to generate pUCBHB1/BoNT/A-XP6A-GRPP.The synthesized polynucleotide molecule is verified by sequencing usingBig Dye Terminator™ Chemistry 3.1 (Applied Biosystems, Foster City,Calif.) and an ABI 3100 sequencer (Applied Biosystems, Foster City,Calif.).

If desired, an expression optimized polynucleotide molecule based onBoNT/A-XP6A-GRPP (SEQ ID NO: 121) can be synthesized in order to improveexpression in an Escherichia coli strain. The polynucleotide moleculeencoding the BoNT/A-XP6A-GRPP will be modified to 1) contain synonymouscodons typically present in native polynucleotide molecules of anEscherichia coli strain; 2) contain a G+C content that more closelymatches the average G+C content of native polynucleotide molecules foundin an Escherichia coli strain; 3) reduce polymononucleotide regionsfound within the polynucleotide molecule; and/or 4) eliminate internalregulatory or structural sites found within the polynucleotide molecule,see, e.g., Lance E. Steward et al., Optimizing Expression of ActiveBotulinum Toxin Type E, International Patent Publication WO 2006/011966(Feb. 2, 2006); Lance E. Steward et al., Optimizing Expression of ActiveBotulinum Toxin Type A, International Patent Publication WO 2006/017749(Feb. 16, 2006). Once sequence optimization is complete,oligonucleotides of 20 to 50 bases in length are synthesized usingstandard phosphoramidite synthesis. These oligonucleotides arehybridized into double stranded duplexes that are ligated together toassemble the full-length polynucleotide molecule. This polynucleotidemolecule is cloned using standard molecular biology methods into apUCBHB1 vector at the SmaI site to generate pUCBHB1/BoNT/A-XP6A-GRPP.The synthesized polynucleotide molecule is verified by sequencing usingBig Dye Terminator™ Chemistry 3.1 (Applied Biosystems, Foster City,Calif.) and an ABI 3100 sequencer (Applied Biosystems, Foster City,Calif.). If so desired, expression optimization to a different organism,such as, e.g., a yeast strain, an insect cell-line or a mammalian cellline, can be done, see, e.g., Steward, supra, (Feb. 2, 2006); andSteward, supra, (Feb. 16, 2006).

A similar cloning strategy will be used to make pUCBHB1 cloningconstructs for BoNT/B-XP6A-GRPP, a modified BoNT/B where amino acids1098-1291 of SEQ ID NO: 2 are replaced with amino acids 21-50 of SEQ IDNO: 9; BoNT/C1-XP6A-GRPP, a modified BoNT/C1 where amino acids 1112-1291of SEQ ID NO: 3 are replaced with amino acids 21-50 of SEQ ID NO: 9;BoNT/D-XP6A-GRPP, a modified BoNT/D where amino acids 1099-1276 of SEQID NO: 4 are replaced with amino acids 21-50 of SEQ ID NO: 9;BoNT/E-XP6A-GRPP, a modified BoNT/E where amino acids 1086-1252 of SEQID NO: 5 are replaced with amino acids 21-50 of SEQ ID NO: 9;BoNT/F-XP6A-GRPP, a modified BoNT/F where amino acids 1106-1274 of SEQID NO: 6 are replaced with amino acids 21-50 of SEQ ID NO: 9;BoNT/G-XP6A-GRPP, a modified BoNT/G where amino acids 1106-1297 of SEQID NO: 7 are replaced with amino acids 21-50 of SEQ ID NO: 9; andTeNT-XP6A-GRPP, a modified TeNT where amino acids 1128-1315 of SEQ IDNO: 8 are replaced with amino acids 21-50 of SEQ ID NO: 9. Likewise, asimilar cloning strategy will be used to make pUCBHB1 cloning constructscomprising a polynucleotide molecule encoding a modified Clostridialtoxin-XP6A with an enhanced targeting domain comprising, e.g, aminoacids 53-81 of SEQ ID NO: 9; amino acids 53-89 of SEQ ID NO: 9; aminoacids 98-124 of SEQ ID NO: 9; amino acids 146-178 of SEQ ID NO: 9; aminoacids 132-158 of SEQ ID NO: 10; amino acids 32-58 of SEQ ID NO: 11;amino acids 32-75 of SEQ ID NO: 11; amino acids 81-107 of SEQ ID NO: 12;amino acids 125-151 of SEQ ID NO: 12; amino acids 81-107 of SEQ ID NO:13; amino acids 124-150 of SEQ ID NO: 13; amino acids 52-78 of SEQ IDNO: 14; amino acids 52-93 of SEQ ID NO: 14; amino acids 28-54 of SEQ IDNO: 15; amino acids 76-92 of SEQ ID NO: 16; amino acids 59-92 of SEQ IDNO: 16; amino acids 41-50 of SEQ ID NO: 17; amino acids 24-50 of SEQ IDNO: 17; amino acids 99-112 of SEQ ID NO: 18; amino acids 159-193 of SEQID NO: 19; amino acids 154-194 of SEQ ID NO: 19; amino acids 35-70 ofSEQ ID NO: 20; amino acids 145-177 of SEQ ID NO: 20; amino acids 1-200of SEQ ID NO: 21; amino acids 1-150 of SEQ ID NO: 22; amino acids 1-202of SEQ ID NO: 23; amino acids 1-201 of SEQ ID NO: 24; amino acids 1-225of SEQ ID NO: 25; amino acids 123-265 of SEQ ID NO: 26; amino acids21-153 of SEQ ID NO: 27; amino acids 57-210 of SEQ ID NO: 28; aminoacids 21-99 of SEQ ID NO: 29; amino acids 31-94 of SEQ ID NO: 29; aminoacids 19-178 of SEQ ID NO: 30; amino acids 1-558 of SEQ ID NO: 31; aminoacids 1-371 of SEQ ID NO: 32; amino acids 49-118 of SEQ ID NO: 33; aminoacids 25-180 of SEQ ID NO: 34; amino acids 1-54 of SEQ ID NO: 35; aminoacids 139-257 of SEQ ID NO: 36; amino acids 129-247 of SEQ ID NO: 37;amino acids 19-257 of SEQ ID NO: 38; amino acids 81-210 of SEQ ID NO:39; amino acids 118-211 of SEQ ID NO: 40; amino acids 107-196 of SEQ IDNO: 41; amino acids 96-197 of SEQ ID NO: 41; amino acids 66-155 of SEQID NO: 42; amino acids 123-218 of SEQ ID NO: 43; amino acids 293-390 ofSEQ ID NO: 44; amino acids 317-414 of SEQ ID NO: 45; amino acids 315-412of SEQ ID NO: 46; amino acids 276-373 of SEQ ID NO: 47; amino acids296-396 of SEQ ID NO: 48; amino acids 370-472 of SEQ ID NO: 49; aminoacids 309-409 of SEQ ID NO: 50; amino acids 323-454 of SEQ ID NO: 51;amino acids 412-513 of SEQ ID NO: 52; amino acids 374-513 of SEQ ID NO:52; amino acids 330-431 of SEQ ID NO: 53; amino acids 293-431 of SEQ IDNO: 53; amino acids 301-402 of SEQ ID NO: 54; amino acids 323-424 of SEQID NO: 55; amino acids 267-372 of SEQ ID NO: 56; amino acids 327-429 ofSEQ ID NO: 57; amino acids 264-364 of SEQ ID NO: 58; amino acids 400-501of SEQ ID NO: 59; amino acids 354-455 of SEQ ID NO: 60; amino acids352-450 of SEQ ID NO: 61; amino acids 281-375 of SEQ ID NO: 62; aminoacids 376-478 of SEQ ID NO: 63; amino acids 313-407 of SEQ ID NO: 64;amino acids 211-308 of SEQ ID NO: 65; amino acids 321-426 of SEQ ID NO:66; amino acids 303-406 of SEQ ID NO: 67; amino acids 247-352 of SEQ IDNO: 68; amino acids 237-352 of SEQ ID NO: 68; amino acids 247-350 of SEQID NO: 69; amino acids 262-366 of SEQ ID NO: 70; or amino acids 233-366of SEQ ID NO: 70.

To construct pET29/BoNT/A-XP6A-GRPP, a pUCBHB1/BoNT/A-XP6A-GRPPconstruct will be digested with restriction endonucleases that 1) willexcise the polynucleotide molecule encoding the open reading frame ofBoNT/A-XP6A-GRPP; and 2) will enable this polynucleotide molecule to beoperably-linked to a pET29 vector (EMD Biosciences-Novagen, Madison,Wis.). This insert will be subcloned using a T4 DNA ligase procedureinto a pET29 vector that is digested with appropriate restrictionendonucleases to yield pET29/BoNT/A-XP6A-GRPP. The ligation mixture willbe transformed into chemically competent E. coli DH5α cells (Invitrogen,Inc, Carlsbad, Calif.) using a heat shock method, will be plated on 1.5%Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of Kanamycin, andwill be placed in a 37° C. incubator for overnight growth. Bacteriacontaining expression constructs will be identified as Kanamycinresistant colonies. Candidate constructs will be isolated using analkaline lysis plasmid mini-preparation procedure and will be analyzedby restriction endonuclease digest mapping to determine the presence andorientation of the insert. This cloning strategy will yield a pET29expression construct comprising the polynucleotide molecule encoding theBoNT/A-XP6A-GRPP operably-linked to a carboxyl terminal polyhistidineaffinity binding peptide.

A similar cloning strategy will be used to make pET29 expressionconstructs for other modified Clostridial toxin-XP6A-GRPP toxins, suchas, e.g., BoNT/B-XP6A-GRPP, BoNT/C1-XP6A-GRPP, BoNT/D-XP6A-GRPP,BoNT/E-XP6A-GRPP, BoNT/F-XP6A-GRPP, BoNT/G-XP6A-GRPP or TeNT-XP6A-GRPP.Likewise, a similar cloning strategy will be used to make pET29expression constructs comprising a polynucleotide molecule encoding amodified Clostridial toxin-XP6A with an enhanced targeting domaincomprising, e.g, amino acids 53-81 of SEQ ID NO: 9; amino acids 53-89 ofSEQ ID NO: 9; amino acids 98-124 of SEQ ID NO: 9; amino acids 146-178 ofSEQ ID NO: 9; amino acids 132-158 of SEQ ID NO: 10; amino acids 32-58 ofSEQ ID NO: 11; amino acids 32-75 of SEQ ID NO: 11; amino acids 81-107 ofSEQ ID NO: 12; amino acids 125-151 of SEQ ID NO: 12; amino acids 81-107of SEQ ID NO: 13; amino acids 124-150 of SEQ ID NO: 13; amino acids52-78 of SEQ ID NO: 14; amino acids 52-93 of SEQ ID NO: 14; amino acids28-54 of SEQ ID NO: 15; amino acids 76-92 of SEQ ID NO: 16; amino acids59-92 of SEQ ID NO: 16; amino acids 41-50 of SEQ ID NO: 17; amino acids24-50 of SEQ ID NO: 17; amino acids 99-112 of SEQ ID NO: 18; amino acids159-193 of SEQ ID NO: 19; amino acids 154-194 of SEQ ID NO: 19; aminoacids 35-70 of SEQ ID NO: 20; amino acids 145-177 of SEQ ID NO: 20;amino acids 1-200 of SEQ ID NO: 21; amino acids 1-150 of SEQ ID NO: 22;amino acids 1-202 of SEQ ID NO: 23; amino acids 1-201 of SEQ ID NO: 24;amino acids 1-225 of SEQ ID NO: 25; amino acids 123-265 of SEQ ID NO:26; amino acids 21-153 of SEQ ID NO: 27; amino acids 57-210 of SEQ IDNO: 28; amino acids 21-99 of SEQ ID NO: 29; amino acids 31-94 of SEQ IDNO: 29; amino acids 19-178 of SEQ ID NO: 30; amino acids 1-558 of SEQ IDNO: 31; amino acids 1-371 of SEQ ID NO: 32; amino acids 49-118 of SEQ IDNO: 33; amino acids 25-180 of SEQ ID NO: 34; amino acids 1-54 of SEQ IDNO: 35; amino acids 139-257 of SEQ ID NO: 36; amino acids 129-247 of SEQID NO: 37; amino acids 19-257 of SEQ ID NO: 38; amino acids 81-210 ofSEQ ID NO: 39; amino acids 118-211 of SEQ ID NO: 40; amino acids 107-196of SEQ ID NO: 41; amino acids 96-197 of SEQ ID NO: 41; amino acids66-155 of SEQ ID NO: 42; amino acids 123-218 of SEQ ID NO: 43; aminoacids 293-390 of SEQ ID NO: 44; amino acids 317-414 of SEQ ID NO: 45;amino acids 315-412 of SEQ ID NO: 46; amino acids 276-373 of SEQ ID NO:47; amino acids 296-396 of SEQ ID NO: 48; amino acids 370-472 of SEQ IDNO: 49; amino acids 309-409 of SEQ ID NO: 50; amino acids 323-454 of SEQID NO: 51; amino acids 412-513 of SEQ ID NO: 52; amino acids 374-513 ofSEQ ID NO: 52; amino acids 330-431 of SEQ ID NO: 53; amino acids 293-431of SEQ ID NO: 53; amino acids 301-402 of SEQ ID NO: 54; amino acids323-424 of SEQ ID NO: 55; amino acids 267-372 of SEQ ID NO: 56; aminoacids 327-429 of SEQ ID NO: 57; amino acids 264-364 of SEQ ID NO: 58;amino acids 400-501 of SEQ ID NO: 59; amino acids 354-455 of SEQ ID NO:60; amino acids 352-450 of SEQ ID NO: 61; amino acids 281-375 of SEQ IDNO: 62; amino acids 376-478 of SEQ ID NO: 63; amino acids 313-407 of SEQID NO: 64; amino acids 211-308 of SEQ ID NO: 65; amino acids 321-426 ofSEQ ID NO: 66; amino acids 303-406 of SEQ ID NO: 67; amino acids 247-352of SEQ ID NO: 68; amino acids 237-352 of SEQ ID NO: 68; amino acids247-350 of SEQ ID NO: 69; amino acids 262-366 of SEQ ID NO: 70; or aminoacids 233-366 of SEQ ID NO: 70.

3b. A Translocation-Enzymatic-Targeting Domain Organization.

A polynucleotide molecule based on BoNT/A-XP6B-GRPP (SEQ ID NO: 122)will be synthesized and cloned into a pUCBHB1 vector as described inExample 1a. This polynucleotide molecule encodes a BoNT/A modified toreplace amino acids 1111-1296 of SEQ ID NO: 1, a BoNT/A H_(CC) targetingdomain, with amino acids 21-50 of SEQ ID NO: 9, a GRPP targeting domain,and has the general domain arrangement of FIG. 6B. If so desired,expression optimization to a different organism, such as, e.g., abacteria, a yeast strain, an insect cell-line or a mammalian cell line,can be done as described above, see, e.g., Steward, supra, (Feb. 2,2006); and Steward, supra, (Feb. 16, 2006).

Likewise, a similar cloning strategy will be used to make pUCBHB1cloning constructs comprising a polynucleotide molecule encoding amodified BoNT/A-XP6B comprising an enhanced targeting domain comprising,e.g, amino acids 53-81 of SEQ ID NO: 9; amino acids 53-89 of SEQ ID NO:9; amino acids 98-124 of SEQ ID NO: 9; amino acids 146-178 of SEQ ID NO:9; amino acids 132-158 of SEQ ID NO: 10; amino acids 32-58 of SEQ ID NO:11; amino acids 32-75 of SEQ ID NO: 11; amino acids 81-107 of SEQ ID NO:12; amino acids 125-151 of SEQ ID NO: 12; amino acids 81-107 of SEQ IDNO: 13; amino acids 124-150 of SEQ ID NO: 13; amino acids 52-78 of SEQID NO: 14; amino acids 52-93 of SEQ ID NO: 14; amino acids 28-54 of SEQID NO: 15; amino acids 76-92 of SEQ ID NO: 16; amino acids 59-92 of SEQID NO: 16; amino acids 41-50 of SEQ ID NO: 17; amino acids 24-50 of SEQID NO: 17; amino acids 99-112 of SEQ ID NO: 18; amino acids 159-193 ofSEQ ID NO: 19; amino acids 154-194 of SEQ ID NO: 19; amino acids 35-70of SEQ ID NO: 20; amino acids 145-177 of SEQ ID NO: 20; amino acids1-200 of SEQ ID NO: 21; amino acids 1-150 of SEQ ID NO: 22; amino acids1-202 of SEQ ID NO: 23; amino acids 1-201 of SEQ ID NO: 24; amino acids1-225 of SEQ ID NO: 25; amino acids 123-265 of SEQ ID NO: 26; aminoacids 21-153 of SEQ ID NO: 27; amino acids 57-210 of SEQ ID NO: 28;amino acids 21-99 of SEQ ID NO: 29; amino acids 31-94 of SEQ ID NO: 29;amino acids 19-178 of SEQ ID NO: 30; amino acids 1-558 of SEQ ID NO: 31;amino acids 1-371 of SEQ ID NO: 32; amino acids 49-118 of SEQ ID NO: 33;amino acids 25-180 of SEQ ID NO: 34; amino acids 1-54 of SEQ ID NO: 35;amino acids 139-257 of SEQ ID NO: 36; amino acids 129-247 of SEQ ID NO:37; amino acids 19-257 of SEQ ID NO: 38; amino acids 81-210 of SEQ IDNO: 39; amino acids 118-211 of SEQ ID NO: 40; amino acids 107-196 of SEQID NO: 41; amino acids 96-197 of SEQ ID NO: 41; amino acids 66-155 ofSEQ ID NO: 42; amino acids 123-218 of SEQ ID NO: 43; amino acids 293-390of SEQ ID NO: 44; amino acids 317-414 of SEQ ID NO: 45; amino acids315-412 of SEQ ID NO: 46; amino acids 276-373 of SEQ ID NO: 47; aminoacids 296-396 of SEQ ID NO: 48; amino acids 370-472 of SEQ ID NO: 49;amino acids 309-409 of SEQ ID NO: 50; amino acids 323-454 of SEQ ID NO:51; amino acids 412-513 of SEQ ID NO: 52; amino acids 374-513 of SEQ IDNO: 52; amino acids 330-431 of SEQ ID NO: 53; amino acids 293-431 of SEQID NO: 53; amino acids 301-402 of SEQ ID NO: 54; amino acids 323-424 ofSEQ ID NO: 55; amino acids 267-372 of SEQ ID NO: 56; amino acids 327-429of SEQ ID NO: 57; amino acids 264-364 of SEQ ID NO: 58; amino acids400-501 of SEQ ID NO: 59; amino acids 354-455 of SEQ ID NO: 60; aminoacids 352-450 of SEQ ID NO: 61; amino acids 281-375 of SEQ ID NO: 62;amino acids 376-478 of SEQ ID NO: 63; amino acids 313-407 of SEQ ID NO:64; amino acids 211-308 of SEQ ID NO: 65; amino acids 321-426 of SEQ IDNO: 66; amino acids 303-406 of SEQ ID NO: 67; amino acids 247-352 of SEQID NO: 68; amino acids 237-352 of SEQ ID NO: 68; amino acids 247-350 ofSEQ ID NO: 69; amino acids 262-366 of SEQ ID NO: 70; or amino acids233-366 of SEQ ID NO: 70.

In addition, similar cloning strategy will be used to produce a modifiedClostridial toxin-XP6B, such as, e.g., BoNT/B-XP6B, BoNT/C1-XP6B,BoNT/D-XP6B, BoNT/E-XP6B, BoNT/F-XP6B, BoNT/G-XP6B or TeNT-XP6B, tocomprise an enhanced targeting domain comprising, e.g., amino acids53-81 of SEQ ID NO: 9; amino acids 53-89 of SEQ ID NO: 9; amino acids98-124 of SEQ ID NO: 9; amino acids 146-178 of SEQ ID NO: 9; amino acids132-158 of SEQ ID NO: 10; amino acids 32-58 of SEQ ID NO: 11; aminoacids 32-75 of SEQ ID NO: 11; amino acids 81-107 of SEQ ID NO: 12; aminoacids 125-151 of SEQ ID NO: 12; amino acids 81-107 of SEQ ID NO: 13;amino acids 124-150 of SEQ ID NO: 13; amino acids 52-78 of SEQ ID NO:14; amino acids 52-93 of SEQ ID NO: 14; amino acids 28-54 of SEQ ID NO:15; amino acids 76-92 of SEQ ID NO: 16; amino acids 59-92 of SEQ ID NO:16; amino acids 41-50 of SEQ ID NO: 17; amino acids 24-50 of SEQ ID NO:17; amino acids 99-112 of SEQ ID NO: 18; amino acids 159-193 of SEQ IDNO: 19; amino acids 154-194 of SEQ ID NO: 19; amino acids 35-70 of SEQID NO: 20; amino acids 145-177 of SEQ ID NO: 20; amino acids 1-200 ofSEQ ID NO: 21; amino acids 1-150 of SEQ ID NO: 22; amino acids 1-202 ofSEQ ID NO: 23; amino acids 1-201 of SEQ ID NO: 24; amino acids 1-225 ofSEQ ID NO: 25; amino acids 123-265 of SEQ ID NO: 26; amino acids 21-153of SEQ ID NO: 27; amino acids 57-210 of SEQ ID NO: 28; amino acids 21-99of SEQ ID NO: 29; amino acids 31-94 of SEQ ID NO: 29; amino acids 19-178of SEQ ID NO: 30; amino acids 1-558 of SEQ ID NO: 31; amino acids 1-371of SEQ ID NO: 32; amino acids 49-118 of SEQ ID NO: 33; amino acids25-180 of SEQ ID NO: 34; amino acids 1-54 of SEQ ID NO: 35; amino acids139-257 of SEQ ID NO: 36; amino acids 129-247 of SEQ ID NO: 37; aminoacids 19-257 of SEQ ID NO: 38; amino acids 81-210 of SEQ ID NO: 39;amino acids 118-211 of SEQ ID NO: 40; amino acids 107-196 of SEQ ID NO:41; amino acids 96-197 of SEQ ID NO: 41; amino acids 66-155 of SEQ IDNO: 42; amino acids 123-218 of SEQ ID NO: 43; amino acids 293-390 of SEQID NO: 44; amino acids 317-414 of SEQ ID NO: 45; amino acids 315-412 ofSEQ ID NO: 46; amino acids 276-373 of SEQ ID NO: 47; amino acids 296-396of SEQ ID NO: 48; amino acids 370-472 of SEQ ID NO: 49; amino acids309-409 of SEQ ID NO: 50; amino acids 323-454 of SEQ ID NO: 51; aminoacids 412-513 of SEQ ID NO: 52; amino acids 374-513 of SEQ ID NO: 52;amino acids 330-431 of SEQ ID NO: 53; amino acids 293-431 of SEQ ID NO:53; amino acids 301-402 of SEQ ID NO: 54; amino acids 323-424 of SEQ IDNO: 55; amino acids 267-372 of SEQ ID NO: 56; amino acids 327-429 of SEQID NO: 57; amino acids 264-364 of SEQ ID NO: 58; amino acids 400-501 ofSEQ ID NO: 59; amino acids 354-455 of SEQ ID NO: 60; amino acids 352-450of SEQ ID NO: 61; amino acids 281-375 of SEQ ID NO: 62; amino acids376-478 of SEQ ID NO: 63; amino acids 313-407 of SEQ ID NO: 64; aminoacids 211-308 of SEQ ID NO: 65; amino acids 321-426 of SEQ ID NO: 66;amino acids 303-406 of SEQ ID NO: 67; amino acids 247-352 of SEQ ID NO:68; amino acids 237-352 of SEQ ID NO: 68; amino acids 247-350 of SEQ IDNO: 69; amino acids 262-366 of SEQ ID NO: 70; or amino acids 233-366 ofSEQ ID NO: 70.

To construct pET29/BoNT/A-XP6B-GRPP, a similar cloning strategy will beused as described in Example 1a. This cloning strategy will yield apET29 expression construct comprising the polynucleotide moleculeencoding the BoNT/A-XP6B-GRPP operably-linked to a carboxyl terminalpolyhistidine affinity binding peptide. A similar cloning strategy willbe used to make pET29 expression constructs for other modifiedClostridial toxin-XP6B-GRPP toxins, such as, e.g., BoNT/B-XP6B-GRPP,BoNT/C1-XP6B-GRPP, BoNT/D-XP6B-GRPP, BoNT/E-XP6B-GRPP, BoNT/F-XP6B-GRPP,BoNT/G-XP6B-GRPP or TeNT-XP6B-GRPP. Likewise, a similar cloning strategywill be used to make pET29 expression constructs comprising apolynucleotide molecule encoding a modified Clostridial toxin-XP6B withan enhanced targeting domain comprising, e.g., amino acids 53-81 of SEQID NO: 9; amino acids 53-89 of SEQ ID NO: 9; amino acids 98-124 of SEQID NO: 9; amino acids 146-178 of SEQ ID NO: 9; amino acids 132-158 ofSEQ ID NO: 10; amino acids 32-58 of SEQ ID NO: 11; amino acids 32-75 ofSEQ ID NO: 11; amino acids 81-107 of SEQ ID NO: 12; amino acids 125-151of SEQ ID NO: 12; amino acids 81-107 of SEQ ID NO: 13; amino acids124-150 of SEQ ID NO: 13; amino acids 52-78 of SEQ ID NO: 14; aminoacids 52-93 of SEQ ID NO: 14; amino acids 28-54 of SEQ ID NO: 15; aminoacids 76-92 of SEQ ID NO: 16; amino acids 59-92 of SEQ ID NO: 16; aminoacids 41-50 of SEQ ID NO: 17; amino acids 24-50 of SEQ ID NO: 17; aminoacids 99-112 of SEQ ID NO: 18; amino acids 159-193 of SEQ ID NO: 19;amino acids 154-194 of SEQ ID NO: 19; amino acids 35-70 of SEQ ID NO:20; amino acids 145-177 of SEQ ID NO: 20; amino acids 1-200 of SEQ IDNO: 21; amino acids 1-150 of SEQ ID NO: 22; amino acids 1-202 of SEQ IDNO: 23; amino acids 1-201 of SEQ ID NO: 24; amino acids 1-225 of SEQ IDNO: 25; amino acids 123-265 of SEQ ID NO: 26; amino acids 21-153 of SEQID NO: 27; amino acids 57-210 of SEQ ID NO: 28; amino acids 21-99 of SEQID NO: 29; amino acids 31-94 of SEQ ID NO: 29; amino acids 19-178 of SEQID NO: 30; amino acids 1-558 of SEQ ID NO: 31; amino acids 1-371 of SEQID NO: 32; amino acids 49-118 of SEQ ID NO: 33; amino acids 25-180 ofSEQ ID NO: 34; amino acids 1-54 of SEQ ID NO: 35; amino acids 139-257 ofSEQ ID NO: 36; amino acids 129-247 of SEQ ID NO: 37; amino acids 19-257of SEQ ID NO: 38; amino acids 81-210 of SEQ ID NO: 39; amino acids118-211 of SEQ ID NO: 40; amino acids 107-196 of SEQ ID NO: 41; aminoacids 96-197 of SEQ ID NO: 41; amino acids 66-155 of SEQ ID NO: 42;amino acids 123-218 of SEQ ID NO: 43; amino acids 293-390 of SEQ ID NO:44; amino acids 317-414 of SEQ ID NO: 45; amino acids 315-412 of SEQ IDNO: 46; amino acids 276-373 of SEQ ID NO: 47; amino acids 296-396 of SEQID NO: 48; amino acids 370-472 of SEQ ID NO: 49; amino acids 309-409 ofSEQ ID NO: 50; amino acids 323-454 of SEQ ID NO: 51; amino acids 412-513of SEQ ID NO: 52; amino acids 374-513 of SEQ ID NO: 52; amino acids330-431 of SEQ ID NO: 53; amino acids 293-431 of SEQ ID NO: 53; aminoacids 301-402 of SEQ ID NO: 54; amino acids 323-424 of SEQ ID NO: 55;amino acids 267-372 of SEQ ID NO: 56; amino acids 327-429 of SEQ ID NO:57; amino acids 264-364 of SEQ ID NO: 58; amino acids 400-501 of SEQ IDNO: 59; amino acids 354-455 of SEQ ID NO: 60; amino acids 352-450 of SEQID NO: 61; amino acids 281-375 of SEQ ID NO: 62; amino acids 376-478 ofSEQ ID NO: 63; amino acids 313-407 of SEQ ID NO: 64; amino acids 211-308of SEQ ID NO: 65; amino acids 321-426 of SEQ ID NO: 66; amino acids303-406 of SEQ ID NO: 67; amino acids 247-352 of SEQ ID NO: 68; aminoacids 237-352 of SEQ ID NO: 68; amino acids 247-350 of SEQ ID NO: 69;amino acids 262-366 of SEQ ID NO: 70; or amino acids 233-366 of SEQ IDNO: 70.

Example 4 Expression of Modified Clostridial Toxins in a Bacterial Cell

The following example illustrates a procedure useful for expressing anyof the modified Clostridial toxins disclosed in the presentspecification in a bacterial cell.

An expression construct, such as, e.g., any of the expression constructsin Examples 1-5, is introduced into chemically competent E. coli BL21(DE3) cells (Invitrogen, Inc, Carlsbad, Calif.) using a heat-shocktransformation protocol. The heat-shock reaction is plated onto 1.5%Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of Kanamycin andis placed in a 37° C. incubator for overnight growth.Kanamycin-resistant colonies of transformed E. coli containing theexpression construct are used to inoculate a baffled flask containing3.0 mL of PA-0.5G media containing 50 μg/mL of Kanamycin which is thenplaced in a 37° C. incubator, shaking at 250 rpm, for overnight growth.The resulting overnight starter culture is in turn used to inoculate a 3L baffled flask containing ZYP-5052 autoinducing media containing 50μg/mL of Kanamycin at a dilution of 1:1000. Culture volumes ranged fromabout 600 mL (20% flask volume) to about 750 mL (25% flask volume).These cultures are grown in a 37° C. incubator shaking at 250 rpm forapproximately 5.5 hours and are then transferred to a 16° C. incubatorshaking at 250 rpm for overnight expression. Cells are harvested bycentrifugation (4,000 rpm at 4° C. for 20-30 minutes) and are usedimmediately, or stored dry at −80° C. until needed.

Example 5 Purification and Quantification of Modified Clostridial Toxins

The following example illustrates methods useful for purification andquantification of any modified Clostridial toxins disclosed in thepresent specification.

For immobilized metal affinity chromatography (IMAC) proteinpurification, E. coli BL21 (DE3) cell pellets used to express a modifiedClostridial toxin, as described in Example 7, are resuspended in ColumnBinding Buffer (25 mM N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonicacid) (HEPES), pH 7.8; 500 mM sodium chloride; 10 mM imidazole; 2×Protease Inhibitor Cocktail Set III (EMD Biosciences-Calbiochem, SanDiego Calif.); 5 units/mL of Benzonase (EMD Biosciences-Novagen,Madison, Wis.); 0.1% (v/v) Triton-X® 100, 4-octylphenol polyethoxylate;10% (v/v) glycerol), and then are transferred to a cold Oakridgecentrifuge tube. The cell suspension is sonicated on ice (10-12 pulsesof 10 seconds at 40% amplitude with 60 seconds cooling intervals on aBranson Digital Sonifier) in order to lyse the cells and then iscentrifuged (16,000 rpm at 4° C. for 20 minutes) to clarify the lysate.An immobilized metal affinity chromatography column is prepared using a20 mL Econo-Pac column support (Bio-Rad Laboratories, Hercules, Calif.)packed with 2.5-5.0 mL of TALON™ SuperFlow Co²⁺ affinity resin (BDBiosciences-Clontech, Palo Alto, Calif.), which is then equilibrated byrinsing with 5 column volumes of deionized, distilled water, followed by5 column volumes of Column Binding Buffer. The clarified lysate isapplied slowly to the equilibrated column by gravity flow (approximately0.25-0.3 mL/minute). The column is then washed with 5 column volumes ofColumn Wash Buffer (N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonicacid) (HEPES), pH 7.8; 500 mM sodium chloride; 10 mM imidazole; 0.1%(v/v) Triton-X® 100, 4-octylphenol polyethoxylate; 10% (v/v) glycerol).The modified Clostridial toxin is eluted with 20-30 mL of Column ElutionBuffer (25 mM N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid)(HEPES), pH 7.8; 500 mM sodium chloride; 500 mM imidazole; 0.1% (v/v)Triton-X® 100, 4-octylphenol polyethoxylate; 10% (v/v) glycerol) and iscollected in approximately twelve 1 mL fractions. The amount of modifiedClostridial toxin contained in each elution fraction is determined by aBradford dye assay. In this procedure, 20 μL aliquots of each 1.0 mLfraction is combined with 200 μL of Bio-Rad Protein Reagent (Bio-RadLaboratories, Hercules, Calif.), diluted 1 to 4 with deionized,distilled water, and then the intensity of the colorimetric signal ismeasured using a spectrophotometer. The five fractions with thestrongest signal are considered the elution peak and are combinedtogether. Total protein yield is determined by estimating the totalprotein concentration of the pooled peak elution fractions using bovinegamma globulin as a standard (Bio-Rad Laboratories, Hercules, Calif.).

For purification of a modified Clostridial toxin using a FPLC desaltingcolumn, a HiPrep™ 26/10 size exclusion column (Amersham Biosciences,Piscataway, N.J.) is pre-equilibrated with 80 mL of 4° C. Column Buffer(50 mM sodium phosphate, pH 6.5). After the column is equilibrated, amodified Clostridial toxin sample is applied to the size exclusioncolumn with an isocratic mobile phase of 4° C. Column Buffer and at aflow rate of 10 mL/minute using a BioLogic DuoFlow chromatography system(Bio-Rad Laboratories, Hercules, Calif.). The desalted modifiedClostridial toxin sample is collected as a single fraction ofapproximately 7-12 mL.

For purification of a modified Clostridial toxin using a FPLC ionexchange column, a modified Clostridial toxin sample that has beendesalted following elution from an IMAC column is applied to a 1 mL Q1™anion exchange column (Bio-Rad Laboratories, Hercules, Calif.) using aBioLogic DuoFlow chromatography system (Bio-Rad Laboratories, Hercules,Calif.). The sample is applied to the column in 4° C. Column Buffer (50mM sodium phosphate, pH 6.5) and is eluted by linear gradient with 4° C.Elution Buffer (50 mM sodium phosphate, 1 M sodium chloride, pH 6.5) asfollows: step 1, 5.0 mL of 5% Elution Buffer at a flow rate of 1mL/minute; step 2, 20.0 mL of 5-30% Elution Buffer at a flow rate of 1mL/minute; step 3, 2.0 mL of 50% Elution Buffer at a flow rate of 1.0mL/minute; step 4, 4.0 mL of 100% Elution Buffer at a flow rate of 1.0mL/minute; and step 5, 5.0 mL of 0% Elution Buffer at a flow rate of 1.0mL/minute. Elution of modified Clostridial toxin from the column ismonitored at 280, 260, and 214 nm, and peaks absorbing above a minimumthreshold (0.01 au) at 280 nm are collected. Most of the modifiedClostridial toxin will elute at a sodium chloride concentration ofapproximately 100 to 200 mM. Average total yields of modifiedClostridial toxin will be determined by a Bradford assay.

Expression of a modified Clostridial toxin is analyzed by polyacrylamidegel electrophoresis. Samples purified using the procedure describedabove are added to 2×LDS Sample Buffer (Invitrogen, Inc, Carlsbad,Calif.) and are separated by MOPS polyacrylamide gel electrophoresisusing NuPAGE® Novex 4-12% Bis-Tris precast polyacrylamide gels(Invitrogen, Inc, Carlsbad, Calif.) under denaturing, reducingconditions. Gels are stained with SYPRO® Ruby (Bio-Rad Laboratories,Hercules, Calif.) and the separated polypeptides are imaged using aFluor-S MAX Multilmager (Bio-Rad Laboratories, Hercules, Calif.) forquantification of modified Clostridial toxin expression levels. The sizeand amount of modified Clostridial toxin is determined by comparison toMagicMark™ protein molecular weight standards (Invitrogen, Inc,Carlsbad, Calif.).

Expression of modified Clostridial toxin is also analyzed by Westernblot analysis. Protein samples purified using the procedure describedabove are added to 2×LDS Sample Buffer (Invitrogen, Inc, Carlsbad,Calif.) and are separated by MOPS polyacrylamide gel electrophoresisusing NuPAGE® Novex 4-12% Bis-Tris precast polyacrylamide gels(Invitrogen, Inc, Carlsbad, Calif.) under denaturing, reducingconditions. Separated polypeptides are transferred from the gel ontopolyvinylidene fluoride (PVDF) membranes (Invitrogen, Inc, Carlsbad,Calif.) by Western blotting using a Trans-Blot® SD semi-dryelectrophoretic transfer cell apparatus (Bio-Rad Laboratories, Hercules,Calif.). PVDF membranes are blocked by incubating at room temperaturefor 2 hours in a solution containing 25 mM Tris-Buffered Saline (25 mM2-amino-2-hydroxymethyl-1,3-propanediol hydrochloric acid (Tris-HCl)(pH7.4), 137 mM sodium chloride, 2.7 mM potassium chloride), 0.1%TWEEN-20®, polyoxyethylene (20) sorbitan monolaureate, 2% bovine serumalbumin, 5% nonfat dry milk. Blocked membranes are incubated at 4° C.for overnight in Tris-Buffered Saline TWEEN-20® (25 mM Tris-BufferedSaline, 0.1% TWEEN-20®, polyoxyethylene (20) sorbitan monolaureate)containing appropriate primary antibodies as a probe. Primary antibodyprobed blots are washed three times for 15 minutes each time inTris-Buffered Saline TWEEN-20®. Washed membranes are incubated at roomtemperature for 2 hours in Tris-Buffered Saline TWEEN-20® containing anappropriate immunoglobulin G antibody conjugated to horseradishperoxidase as a secondary antibody. Secondary antibody-probed blots arewashed three times for 15 minutes each time in Tris-Buffered SalineTWEEN-20®. Signal detection of the labeled modified Clostridial toxinare visualized using the ECL Plus™ Western Blot Detection System(Amersham Biosciences, Piscataway, N.J.) and are imaged with a Typhoon9410 Variable Mode Imager (Amersham Biosciences, Piscataway, N.J.) forquantification of modified Clostridial toxin expression levels.

Although aspects of the present invention have been described withreference to the disclosed embodiments, one skilled in the art willreadily appreciate that the specific examples disclosed are onlyillustrative of these aspects and in no way limit the present invention.Various modifications can be made without departing from the spirit ofthe present invention.

What is claimed is:
 1. A modified Clostridial toxin comprising: a) aClostridial toxin enzymatic domain capable of executing an enzymatictarget modification step of a Clostridial toxin intoxication process; b)a Clostridial toxin translocation domain capable of executing atranslocation step of a Clostridial toxin intoxication process; c) atargeting domain comprising a polypeptide consisting of an axon guidancesignaling molecule, such that the targeting domain is capable ofexecuting a cell binding step of a Clostridial toxin intoxicationprocess; and d) a protease cleavage site, wherein cleavage of theprotease cleavage site converts the single-chain form of the modifiedClostridial toxin into the di-chain form.
 2. The modified Clostridialtoxin according to claim 1, wherein the modified Clostridial toxincomprises, in a linear amino-to-carboxyl single polypeptide order, theClostridial toxin enzymatic domain, the protease cleavage site, theClostridial toxin translocation domain and the targeting domain.
 3. Themodified Clostridial toxin according to claim 1, wherein the modifiedClostridial toxin comprises, in a linear amino-to-carboxyl singlepolypeptide order, the Clostridial toxin enzymatic domain, the proteasecleavage site, the targeting domain and the Clostridial toxintranslocation domain.
 4. The modified Clostridial toxin according toclaim 1, wherein the modified Clostridial toxin comprises, in a linearamino-to-carboxyl single polypeptide order, the targeting domain, theClostridial toxin translocation domain, the protease cleavage site andthe Clostridial toxin enzymatic domain.
 5. The modified Clostridialtoxin according to claim 1, wherein the modified Clostridial toxincomprises, in a linear amino-to-carboxyl single polypeptide order, thetargeting domain, the Clostridial toxin enzymatic domain, the proteasecleavage site and the Clostridial toxin translocation domain.
 6. Themodified Clostridial toxin according to claim 1, wherein the modifiedClostridial toxin comprises, in a linear amino-to-carboxyl singlepolypeptide order, the Clostridial toxin translocation domain, theprotease cleavage site, the Clostridial toxin enzymatic domain and thetargeting domain.
 7. The modified Clostridial toxin according to claim1, wherein the modified Clostridial toxin comprises, in a linearamino-to-carboxyl single polypeptide order, the Clostridial toxintranslocation domain, the protease cleavage site, the targeting domainand the Clostridial toxin enzymatic domain.
 8. The modified Clostridialtoxin according to claim 1, wherein the Clostridial toxin enzymaticdomain is selected from the group consisting of a BoNT/A enzymaticdomain, a BoNT/B enzymatic domain, a BoNT/C1 enzymatic domain, a BoNT/Denzymatic domain, a BoNT/E enzymatic domain, a BoNT/F enzymatic domain,a BoNT/G enzymatic domain and a TeNT enzymatic domain.
 9. The modifiedClostridial toxin according to claim 1, wherein the Clostridial toxintranslocation domain is selected from the group consisting of a BoNT/Atranslocation domain, a BoNT/B translocation domain, a BoNT/C1translocation domain, a BoNT/D translocation domain, a BoNT/Etranslocation domain, a BoNT/F translocation domain, a BoNT/Gtranslocation domain and a TeNT translocation domain.
 10. The modifiedClostridial toxin according to claim 1, wherein the protease cleavagesite is an endogenous Clostridial toxin di-chain loop protease cleavagesite or an exogenous cleavage site.
 11. The modified Clostridial toxinaccording to claim 10, wherein the endogenous Clostridial toxin di-chainloop protease cleavage site is selected from the group consisting of aBoNT/A di-chain loop protease cleavage site, a BoNT/B di-chain loopprotease cleavage site, a BoNT/C1 di-chain loop protease cleavage site,a BoNT/D di-chain loop protease cleavage site, a BoNT/E di-chain loopprotease cleavage site, a BoNT/F di-chain loop protease cleavage site, aBoNT/G di-chain loop protease cleavage site and a TeNT di-chain loopprotease cleavage site.
 12. The modified Clostridial toxin according toclaim 10, wherein the exogenous protease cleavage site is selected fromthe group consisting of an enterokinase cleavage site, a Thrombincleavage site, a Factor Xa cleavage site, a human rhinovirus 3C proteasecleavage site, a tobacco etch virus protease cleavage site, a dipeptidylaminopeptidase cleavage site, a small ubiquitin-like modifier(SUMO)/ubiquitin-like protein-1(ULP-1) protease cleavage site, and aClostridial toxin substrate cleavage site.
 13. The modified Clostridialtoxin according to claim 12, wherein the Clostridial toxin substratecleavage site is selected from the group consisting of a BoNT/Asubstrate cleavage site, a BoNT/B substrate cleavage site, a BoNT/C1substrate cleavage site, a BoNT/D substrate cleavage site, a BoNT/Esubstrate cleavage site, a BoNT/F substrate cleavage site, a BoNT/Gsubstrate cleavage site and a TeNT substrate cleavage site.